MSTN recombinant vaccine and application

Abstract

The invention relates to the technical field of biology, in particular to an MSTN recombinant vaccine and application. The invention provides the MSTN recombinant protein, the antigen purity is high, the safety is good, the MSTN recombinant protein is used for animal immunization, the prepared MSTN recombinant vaccine has no pathogenicity to animals such as mice, the safety evaluation is easy to pass, meanwhile, the vaccine has an efficient antigen presentation mode, an immune system can be effectively induced to generate immune protection response, animal muscle growth is stimulated, and the immune protection effect is good. The important application prospect is realized in the field of prevention and treatment of related diseases such as amyotrophy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an MSTN recombinant vaccine and its application. Background Art

[0002] Myostatin (MSTN), also known as GDF-8 (growth differentiation factor 8), is a member of the transforming growth factor-β (TGF-β) family, mainly secreted by skeletal muscle, and plays a negative regulatory role in the process of muscle growth and development. In 1997, American scholars McPherron et al. first cloned the MSTN gene and proved its function. Techniques such as the CRISP / Cas9 system, RNA interference, and anti-MSTN antibodies can be used to knock out or inhibit the expression of the MSTN gene, which can increase the muscle weight of the body and improve muscle function. In animals, after MSTN loses its inhibitory function, the skeletal muscle of vertebrates such as cattle, pigs, and sheep can proliferate significantly, resulting in a "double-muscle" phenotype. For example, the double-muscle buttocks phenomenon of Belgian Blue cattle and Piedmontese cattle caused by natural mutations has a much higher meat production rate than other breeds of cattle, further proving the function of the MSTN gene. Currently, this breed of cattle has been promoted to more than 20 countries. Therefore, the livestock and poultry breeding method based on the modification of the MSTN gene has been widely applied and promoted by scholars. In the treatment of human diseases, for human muscle atrophy-related diseases, such as muscle atrophy or loss caused by Becker muscular dystrophy, Duchenne muscular dystrophy, spinal muscular atrophy, pancreatic cancer, advanced kidney disease, etc., anti-MSTN monoclonal antibodies or affinity substances have been widely studied, and more than 10 corresponding biological drugs have entered clinical trials. There are also experiments showing that MSTN monoclonal antibodies or affinity substances can treat the obesity symptoms in the later stage of type 2 diabetes patients, and the related drugs have entered the clinical trial stage.

[0003] Before MSTN takes effect, it exists in an inactive state. It consists of a precursor protein composed of an N-terminal region and a C-terminal region. There is a protease cleavage site between the two terminal regions. The protease cleavage site of human MSTN is at amino acid position 266. After protease hydrolysis, it forms two parts: an N-terminal protein and a C-terminal protein, but the two still exist in the form of a conjugate in the blood and are inactive. It is not until the 75th aspartic acid of the N-terminal protein is cleaved by a metalloprotease family enzyme that the C-terminal protein can be released to form an active C-terminal protein, which plays a role in inhibiting skeletal muscle protein synthesis.

[0004] MSTN consists of 375 amino acids. Among different species, the homology of MSTN is relatively high, especially in the C-terminal active region. The C-terminal active regions of MSTN in mice, pigs, dogs, cats, and chickens are the same as those in humans, and those in cattle, goats, and sheep are more than 95%. There are also other proteins that inhibit skeletal muscle synthesis, such as GDF-11, and its principle is similar to that of MSTN.

[0005] Currently, in the aspect of livestock and poultry, the research on the MSTN target mainly focuses on livestock and poultry breeding. It mainly uses natural hybridization to screen the offspring with MSTN gene mutations, but the screening time by natural hybridization is relatively long, and the genetic stability of the offspring remains to be verified. If genetic engineering means are used to mutate the MSTN gene, there is a risk of transgenic organism safety.

[0006] In addition, in the aspect of diseases related to human muscle atrophy, more research has been done on using antibodies or affinity substances against MSTN. However, MSTN antibodies or their affinity substances have problems such as short half-life, rejection effect, high production cost, and the need for frequent injection. Although more than a dozen related drugs have entered clinical trials, no related drugs have been approved yet.

[0007] In the research on MSTN vaccines, since MSTN is a protein of the body itself, directly immunizing with MSTN is difficult to cause the body's own immune response. Some scholars have prepared vaccines by mutating the MSTN protein or adding T cell epitopes, but the immunogenicity of the prepared vaccines is poor. Some scholars have also studied MSTN polypeptide vaccines, but the polypeptide vaccines have a single target and also have the disadvantage of poor immunogenicity. There are also reports on the development of MSTN nucleic acid vaccines, but the production cost of nucleic acid vaccines is high, they are not convenient to use, the injected MSTN nucleic acid has the risk of integrating into the body, and the MSTN expressed by nucleic acid vaccines is also similar in structure to natural MSTN and is difficult to stimulate the body's own immune response. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide an MSTN recombinant vaccine and its application.

[0009] The present invention provides a recombinant protein, which comprises at least one of virus-like particle protein, nanoparticle protein, Fc or MBP and MSTN;

[0010] The virus-like particle protein comprises AP205, Qβ, MS2, T4, TMV, CPMV, CMV, PapMV, FHV, HBsAg and / or IFUV;

[0011] The nanoparticle protein comprises Ferritin;

[0012] In the present invention, the source of the MSTN includes humans or other animals, specifically, such as dogs, cats, pigs, chickens, etc., or sequences with an amino acid sequence homology of more than 90%, and the present invention does not limit this.

[0013] In the present invention, the number of the MSTN can be n, where n is an integer between 1 and 10. Specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The n MSTNs can be connected by a linker, or by chemical coupling, or directly chemically synthesized. The present invention does not limit this. The linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10. Specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0014] In the present invention, at least one of the virus-like particle protein, nanoparticle protein, Fc, or MBP can be located at the N-terminus of the MSTN or at the C-terminus of the MSTN. In some specific embodiments of the present invention, at least one of the virus-like particle protein, nanoparticle protein, Fc, or MBP is located at the C-terminus of the MSTN. In other embodiments of the present invention, at least one of the virus-like particle protein, nanoparticle protein, Fc, or MBP is located at the N-terminus of the MSTN.

[0015] Furthermore, in the MSTN recombinant protein of the present invention, the MSTN has the following amino acid sequence:

[0016] (1) The amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or

[0017] (2) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (1); or

[0018] (3) A sequence with a homology of more than 90% to the amino acid sequence shown in (1);

[0019] In the present invention, the MSTN with the amino acid sequence shown in SEQ ID NO: 1 is the natural MSTN protein, abbreviated as W-MSTN; the mutant MSTN obtained by amino acid sequence mutation (D75A, R263S, R266A) based on the amino acid sequence shown in SEQ ID NO: 1 is abbreviated as M-MSTN, and its amino acid sequence is shown in SEQ ID NO: 2; the MSTN with the amino acid sequence shown in SEQ ID NO: 3 is the C-terminal active region of MSTN, abbreviated as C-MSTN, which is the amino acid segment at positions 267-375 of MSTN.

[0020] Specifically, the amino acid sequence of the W-MSTN protein is:

[0021]

[0022] The amino acid sequence of the M-MSTN protein is:

[0023]

[0024]

[0025] The amino acid sequence of the C-MSTN is:

[0026]

[0027] The preparation methods of the W-MSTN, M-MSTN, and C-MSTN include: biosynthesis and / or solid-phase synthesis. The biosynthesis method is: cloning the nucleotide sequences corresponding to the amino acid sequences of W-MSTN, M-MSTN, and C-MSTN into an expression vector to obtain a recombinant expression vector, and then transferring the recombinant expression vector into a host for expression; the solid-phase synthesis method is to synthesize the W-MSTN, M-MSTN, and C-MSTN with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 respectively by using solid-phase synthesis technology.

[0028] In a specific embodiment of the present invention, the MSTN recombinant protein is a W-MSTN-AP205 recombinant protein, an M-MSTN-AP205 recombinant protein or a C-MSTN-AP205 recombinant protein, and the AP205 is derived from the capsid of AP205 phage. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the AP205 is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0029] The amino acid sequence of the AP205 includes:

[0030]

[0031] Or a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 4;

[0032] Or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 4.

[0033] In the present invention, the preparation method of the AP205 includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding AP205 into an expression vector, and obtaining it by expression after transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize AP205 with an amino acid sequence as shown in SEQ ID NO: 4 by using solid-phase synthesis technology.

[0034] The present invention provides a method for preparing recombinant proteins of W-MSTN-AP205, M-MSTN-AP205 or C-MSTN-AP205, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding AP205 into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the recombinant proteins of W-MSTN-AP205, M-MSTN-AP205 or C-MSTN-AP205 are obtained by expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding AP205 by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS. The flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of the recombinant proteins of W-MSTN-AP205, M-MSTN-AP205 or C-MSTN-AP205 by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with AP205 respectively to obtain the recombinant proteins of W-MSTN-AP205, M-MSTN-AP205 or C-MSTN-AP205, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with AP205 respectively to obtain the recombinant proteins of W-MSTN-AP205, M-MSTN-AP205 or C-MSTN-AP205.

[0035] Correspondingly, the present invention also provides:

[0036] I), nucleic acids encoding the MSTN recombinant proteins;

[0037] II), expression units containing the nucleic acids as described in I);

[0038] III), recombinant vectors containing the nucleic acids as described in I or the expression units as described in II;

[0039] IV), host cells transformed or transfected with the recombinant vectors as described in III;

[0040] V), culture products of the host cells as described in IV;

[0041] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0042] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the use of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The immunization dose administered is 25 μg, 50 μg, 100 μg, and / or 200 μg. After immunization for 14 to 100 days, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grasping force increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0043] In specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-Qβ recombinant protein, an M-MSTN-Qβ recombinant protein, or a C-MSTN-Qβ recombinant protein, and the Qβ is from the Qβ phage capsid. The number of W-MSTN, M-MSTN, or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the n W-MSTN, M-MSTN, or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the Qβ is located at the N-terminus or C-terminus of W-MSTN, M-MSTN, or C-MSTN.

[0044] The amino acid sequence of the Qβ includes:

[0045]

[0046] or a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 5;

[0047] or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 5.

[0048] In the present invention, the preparation method of the Qβ includes solid-phase synthesis and / or biosynthesis; the biosynthesis is as follows: a recombinant expression vector is obtained by cloning the nucleic acid encoding Qβ into an expression vector, and after transferring the recombinant expression vector into a host, expression is carried out. The solid-phase synthesis is to synthesize Qβ with an amino acid sequence as shown in SEQ ID NO: 5 by using solid-phase synthesis technology.

[0049] The present invention provides a preparation method of W-MSTN-Qβ, M-MSTN-Qβ or C-MSTN-Qβ recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding Qβ into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, W-MSTN-Qβ, M-MSTN-Qβ or C-MSTN-Qβ recombinant protein is obtained by expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding Qβ by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-Qβ, M-MSTN-Qβ or C-MSTN-Qβ recombinant protein by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with Qβ respectively to obtain W-MSTN-Qβ, M-MSTN-Qβ or C-MSTN-Qβ recombinant protein, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with Qβ respectively to obtain W-MSTN-Qβ, M-MSTN-Qβ or C-MSTN-Qβ recombinant protein.

[0050] Correspondingly, the present invention also provides:

[0051] I), a nucleic acid encoding an MSTN recombinant protein;

[0052] II), an expression unit containing the nucleic acid as described in I);

[0053] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0054] IV), transforming or transfecting a host cell with the recombinant vector as described in III);

[0055] V), the culture product of the host cell as described in IV);

[0056] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0057] Immunizing an animal with this vaccine can obtain the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the use of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The administered immunization dose is 25 μg, 50 μg, 100 μg, and / or 200 μg. 14 to 100 days after immunization, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0058] In a specific embodiment of the present invention, the MSTN recombinant protein is a W-MSTN-MS2 recombinant protein, an M-MSTN-MS2 recombinant protein or a C-MSTN-MS2 recombinant protein, and the MS2 is from the MS2 phage capsid. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the MS2 is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0059] The amino acid sequence of the MS2 includes:

[0060] The amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;

[0061] Or a sequence in which one or more amino acids are substituted, deleted, added, and / or replaced on the basis of the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;

[0062] Or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0063] The MS2 includes a coat protein and a maturase protein, and the coat protein has the amino acid sequence shown below:

[0064]

[0065] The maturase protein has the amino acid sequence shown below:

[0066]

[0067]

[0068] In the present invention, the preparation method of the MS2 includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding MS2 into an expression vector, and obtaining the MS2 by expression after transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize MS2 with an amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7 by using solid-phase synthesis technology.

[0069] The present invention provides a method for preparing W-MSTN-MS2, M-MSTN-MS2 or C-MSTN-MS2 recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding MS2 into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the W-MSTN-MS2, M-MSTN-MS2 or C-MSTN-MS2 recombinant protein is obtained through expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding MS2 by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-MS2, M-MSTN-MS2 or C-MSTN-MS2 recombinant protein by using solid-phase synthesis technology; the chemical coupling includes obtaining W-MSTN-MS2, M-MSTN-MS2 or C-MSTN-MS2 recombinant protein by chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with MS2 respectively, or obtaining W-MSTN-MS2, M-MSTN-MS2 or C-MSTN-MS2 recombinant protein by chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with MS2 respectively.

[0070] Correspondingly, the present invention also provides:

[0071] I), nucleic acid encoding the MSTN recombinant protein;

[0072] II), an expression unit containing the nucleic acid as described in I);

[0073] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0074] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0075] V), a culture product of the host cell as described in IV);

[0076] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0077] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The administered immunization dose is 25 μg, 50 μg, 100 μg, and / or 200 μg. After immunization for 14 to 100 days, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat rate decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0078] In specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-T4 recombinant protein, an M-MSTN-T4 recombinant protein or a C-MSTN-T4 recombinant protein, and the T4 is from the T4 phage capsid. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the T4 is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0079] The amino acid sequence of the T4 includes:

[0080] The amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9;

[0081] or a sequence obtained by substitution, deletion, addition, and / or replacement of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9;

[0082] or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9.

[0083] T4 includes the outer shell proteins Soc and Hoc; the Soc has the amino acid sequence shown as follows:

[0084] MASTRGYVNIKTFEQKLDGNKKIEGKEISVAFPLYSDVHKISGAHYQTFPSEKAAYSTVYEENQRTEWIAANEDLWKVTG (SEQ ID NO: 8);

[0085] Hoc has the amino acid sequence shown as follows:

[0086]

[0087] In the present invention, the preparation method of the T4 includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding T4 into an expression vector, and obtaining it by expression after transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize T4 with an amino acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 9 by using solid-phase synthesis technology.

[0088] The present invention provides a method for preparing W-MSTN-T4, M-MSTN-T4 or C-MSTN-T4 recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding T4 into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the W-MSTN-T4, M-MSTN-T4 or C-MSTN-T4 recombinant protein is obtained through expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding T4 by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS. The flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-T4, M-MSTN-T4 or C-MSTN-T4 recombinant protein by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with T4 respectively to obtain W-MSTN-T4, M-MSTN-T4 or C-MSTN-T4 recombinant protein, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with T4 respectively to obtain W-MSTN-T4, M-MSTN-T4 or C-MSTN-T4 recombinant protein.

[0089] Correspondingly, the present invention also provides:

[0090] I), nucleic acid encoding the MSTN recombinant protein;

[0091] II), an expression unit containing the nucleic acid as described in I);

[0092] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0093] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0094] V), a culture product of the host cell as described in IV);

[0095] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0096] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the use of the above vaccine in increasing muscle, enhancing strength, or reducing body fat in certain diseases in humans or animals. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The administered immunization dose is 25 μg, 50 μg, 100 μg, and / or 200 μg. 14 to 100 days after immunization, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grasping force increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0097] In specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-TMV recombinant protein, an M-MSTN-TMV recombinant protein, or a C-MSTN-TMV recombinant protein, and the TMV is from tobacco mosaic virus. The number of W-MSTN, M-MSTN, or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the n W-MSTN, M-MSTN, or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the TMV is located at the N-terminus or C-terminus of W-MSTN, M-MSTN, or C-MSTN.

[0098] The amino acid sequence of the TMV includes:

[0099]

[0100] or a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 10;

[0101] or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 10.

[0102] In the present invention, the preparation method of the TMV includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding the TMV into an expression vector, and obtaining the TMV after expression by transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize the TMV with an amino acid sequence as shown in SEQ ID NO: 10 by using solid-phase synthesis technology.

[0103] The present invention provides a preparation method of the recombinant protein of W-MSTN-TMV, M-MSTN-TMV or C-MSTN-TMV. The preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding the TMV into an expression vector separately or in a fused form to obtain a recombinant expression vector, and obtain the recombinant protein of W-MSTN-TMV, M-MSTN-TMV or C-MSTN-TMV after expression by transferring the recombinant expression vector into a host. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding the TMV by using the nucleic acid encoding a linker. The sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of the recombinant protein of W-MSTN-TMV, M-MSTN-TMV or C-MSTN-TMV by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with the TMV respectively to obtain the recombinant protein of W-MSTN-TMV, M-MSTN-TMV or C-MSTN-TMV, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with the TMV respectively to obtain the recombinant protein of W-MSTN-TMV, M-MSTN-TMV or C-MSTN-TMV.

[0104] Correspondingly, the present invention also provides:

[0105] I), the nucleic acid encoding the MSTN recombinant protein;

[0106] II), an expression unit containing the nucleic acid as described in I);

[0107] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0108] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0109] V), a culture product of the host cell as described in IV);

[0110] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0111] Immunizing animals with this vaccine can obtain the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of a conjugate vaccine, including administering the vaccine as described above. The administered immunization dose is 25 μg, 50 μg, 100 μg, and / or 200 μg. After immunization for 14 to 100 days, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0112] In the specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-CPMV recombinant protein, an M-MSTN-CPMV recombinant protein or a C-MSTN-CPMV recombinant protein, and the CPMV is derived from cowpea mosaic virus. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the CPMV is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0113] The amino acid sequence of the CPMV includes:

[0114]

[0115] Or a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 11;

[0116] Or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 11.

[0117] In the present invention, the preparation method of the CPMV includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: a recombinant expression vector is obtained by cloning the nucleic acid encoding CPMV into an expression vector, and the recombinant expression vector is transferred into a host and then obtained through expression. The solid-phase synthesis is to synthesize CPMV with an amino acid sequence as shown in SEQ ID NO: 11 by using solid-phase synthesis technology.

[0118] The present invention provides a method for preparing W-MSTN-CPMV, M-MSTN-CPMV or C-MSTN-CPMV recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding CPMV into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the W-MSTN-CPMV, M-MSTN-CPMV or C-MSTN-CPMV recombinant protein is obtained through expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding CPMV by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-CPMV, M-MSTN-CPMV or C-MSTN-CPMV recombinant protein by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with CPMV respectively to obtain W-MSTN-CPMV, M-MSTN-CPMV or C-MSTN-CPMV recombinant protein, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with CPMV respectively to obtain W-MSTN-CPMV, M-MSTN-CPMV or C-MSTN-CPMV recombinant protein.

[0119] Correspondingly, the present invention also provides:

[0120] I), nucleic acid encoding the MSTN recombinant protein;

[0121] II), an expression unit containing the nucleic acid as described in I);

[0122] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0123] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0124] V), the culture product of the host cell as described in IV);

[0125] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0126] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The immunization dose administered is 25 μg, 50 μg, 100 μg, and / or 200 μg. After immunization for 14 to 100 days, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grasping force increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0127] In the specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-CMV recombinant protein, an M-MSTN-CMV recombinant protein or a C-MSTN-CMV recombinant protein, and the CMV is from cucumber mosaic virus. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the CMV is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0128] The amino acid sequence of the CMV includes:

[0129]

[0130] or a sequence with substitution, deletion, addition and / or replacement of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 12;

[0131] or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 12.

[0132] In the present invention, the preparation method of the CMV includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding CMV into an expression vector, and obtaining it after expression by transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize CMV with an amino acid sequence as shown in SEQ ID NO: 12 by using solid-phase synthesis technology.

[0133] The present invention provides a preparation method of W-MSTN-CMV, M-MSTN-CMV or C-MSTN-CMV recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding CMV into an expression vector separately or in a fused form to obtain a recombinant expression vector, and obtain W-MSTN-CMV, M-MSTN-CMV or C-MSTN-CMV recombinant protein after expression by transferring the recombinant expression vector into a host. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding CMV by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-CMV, M-MSTN-CMV or C-MSTN-CMV recombinant protein by using solid-phase synthesis technology; the chemical coupling includes obtaining W-MSTN-CMV, M-MSTN-CMV or C-MSTN-CMV recombinant protein by chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with CMV respectively, or obtaining W-MSTN-CMV, M-MSTN-CMV or C-MSTN-CMV recombinant protein by chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with CMV respectively.

[0134] Correspondingly, the present invention also provides:

[0135] I), the nucleic acid encoding the MSTN recombinant protein;

[0136] II), an expression unit containing the nucleic acid as described in I);

[0137] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0138] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0139] V), a culture product of the host cell as described in IV);

[0140] The present invention also provides a vaccine containing the MSTN recombinant protein. An adjuvant is also included in the vaccine. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0141] Immunizing an animal with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The immunization dose administered is 25 μg, 50 μg, 100 μg, and / or 200 μg. 14 to 100 days after immunization, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grasping force increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat rate decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0142] In a specific embodiment of the present invention, the MSTN recombinant protein is a W-MSTN-PapMV recombinant protein, an M-MSTN-PapMV recombinant protein or a C-MSTN-PapMV recombinant protein, and the PapMV is from papaya mosaic virus. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the PapMV is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0143] The amino acid sequence of the PapMV includes:

[0144]

[0145] or a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 13;

[0146] or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 13.

[0147] In the present invention, the preparation method of the PapMV includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding PapMV into an expression vector, and obtaining it after expression by transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize PapMV with an amino acid sequence as shown in SEQ ID NO: 13 by using solid-phase synthesis technology.

[0148] The present invention provides a method for preparing W-MSTN-PapMV, M-MSTN-PapMV or C-MSTN-PapMV recombinant proteins, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding PapMV into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the W-MSTN-PapMV, M-MSTN-PapMV or C-MSTN-PapMV recombinant protein is obtained through expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding PapMV by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-PapMV, M-MSTN-PapMV or C-MSTN-PapMV recombinant protein by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with PapMV respectively to obtain W-MSTN-PapMV, M-MSTN-PapMV or C-MSTN-PapMV recombinant protein, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with PapMV respectively to obtain W-MSTN-PapMV, M-MSTN-PapMV or C-MSTN-PapMV recombinant protein.

[0149] Correspondingly, the present invention also provides:

[0150] I), nucleic acid encoding the MSTN recombinant protein;

[0151] II), an expression unit containing the nucleic acid as described in I);

[0152] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0153] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0154] V), a culture product of the host cell as described in IV);

[0155] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the W-MSTN-PapMV, M-MSTN-PapMV or C-MSTN-PapMV recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0156] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the use of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The immunization dose administered is 25 μg, 50 μg, 100 μg and / or 200 μg. 14 to 100 days after immunization, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0157] In specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-FHV recombinant protein, an M-MSTN-FHV recombinant protein or a C-MSTN-FHV recombinant protein, and the FHV is from the shed virus. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the FHV is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0158] The amino acid sequence of the FHV includes:

[0159]

[0160]

[0161] or a sequence obtained by substitution, deletion, addition, and / or replacement of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 14;

[0162] or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 14.

[0163] In the present invention, the preparation method of the FHV includes solid-phase synthesis and / or biosynthesis; the biosynthesis is obtained by cloning the nucleic acid encoding FHV into an expression vector to obtain a recombinant expression vector, and then expressing the recombinant expression vector in a host. The solid-phase synthesis is to synthesize the FHV with the amino acid sequence shown in SEQ ID NO: 14 by using solid-phase synthesis technology.

[0164] The present invention provides a preparation method of W-MSTN-FHV, M-MSTN-FHV or C-MSTN-FHV recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding FHV into an expression vector separately or in a fused form to obtain a recombinant expression vector, and then express the recombinant expression vector in a host to obtain W-MSTN-FHV, M-MSTN-FHV or C-MSTN-FHV recombinant protein. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding AP205 by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-FHV, M-MSTN-FHV or C-MSTN-FHV recombinant protein by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with FHV respectively to obtain W-MSTN-FHV, M-MSTN-FHV or C-MSTN-FHV recombinant protein, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with FHV respectively to obtain W-MSTN-FHV, M-MSTN-FHV or C-MSTN-FHV recombinant protein.

[0165] Correspondingly, the present invention also provides:

[0166] I), the nucleic acid encoding the MSTN recombinant protein;

[0167] II), an expression unit containing the nucleic acid as described in I);

[0168] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0169] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0170] V), a culture product of the host cell as described in IV);

[0171] The present invention also provides a vaccine of the MSTN recombinant protein. An adjuvant is also included in the vaccine. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0172] Immunizing an animal with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the use of the above vaccine in increasing muscle, enhancing strength or reducing body fat in certain diseases in humans or animals. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The immunization dose administered is 25 μg, 50 μg, 100 μg and / or 200 μg. 14 to 100 days after immunization, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grasping force increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0173] In a specific embodiment of the present invention, the MSTN recombinant protein is a W-MSTN-HBsAg recombinant protein, an M-MSTN-HBsAg recombinant protein or a C-MSTN-HBsAg recombinant protein, and the HBsAg is derived from hepatitis B surface antigen. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the HBsAg is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0174] The amino acid sequence of the HBsAg includes:

[0175]

[0176] Or a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 15;

[0177] Or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 15.

[0178] In the present invention, the preparation method of the HBsAg includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding HBSAG into an expression vector, and obtaining it by expression after transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize HBsAg with an amino acid sequence as shown in SEQ ID NO: 15 by using solid-phase synthesis technology.

[0179] The present invention provides a method for preparing recombinant proteins of W-MSTN-HBsAg, M-MSTN-HBsAg or C-MSTN-HBsAg, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding HBsAg into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the recombinant proteins of W-MSTN-HBsAg, M-MSTN-HBsAg or C-MSTN-HBsAg are obtained through expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding HBsAg by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of the recombinant proteins of W-MSTN-HBsAg, M-MSTN-HBsAg or C-MSTN-HBsAg by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with HBsAg respectively to obtain the recombinant proteins of W-MSTN-HBsAg, M-MSTN-HBsAg or C-MSTN-HBsAg, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with HBsAg respectively to obtain the recombinant proteins of W-MSTN-HBsAg, M-MSTN-HBsAg or C-MSTN-HBsAg

[0180] Correspondingly, the present invention also provides:

[0181] I), the nucleic acid encoding the MSTN recombinant protein;

[0182] II), an expression unit containing the nucleic acid as described in I);

[0183] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0184] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0185] V), the culture product of the host cell as described in IV);

[0186] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0187] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The administered immunization doses are 25 μg, 50 μg, 100 μg, and / or 200 μg. After immunization for 14 to 100 days, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat rate decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0188] In specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-IFUV recombinant protein, an M-MSTN-IFUV recombinant protein, or a C-MSTN-IFUV recombinant protein, and the IFUV is derived from the influenza matrix M1 protein. The number of W-MSTN, M-MSTN, or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the n W-MSTN, M-MSTN, or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the IFUV is located at the N-terminus or C-terminus of W-MSTN, M-MSTN, or C-MSTN.

[0189] The IFUV is a virus-like particle formed by the co-expression of the influenza virus M1 matrix protein and the HA protein. The amino acid sequence of M1 is:

[0190]

[0191] The amino acid sequence of HA is:

[0192]

[0193] The amino acid sequence of the IFUV includes:

[0194] The amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17;

[0195] Or a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17;

[0196] Or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17.

[0197] In the present invention, the preparation method of the IFUV includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding the IFUV into an expression vector, and obtaining it by expression after transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize the IFUV with an amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17 by using solid-phase synthesis technology.

[0198] The present invention provides a method for preparing W-MSTN-IFUV, M-MSTN-IFUV or C-MSTN-IFUV recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding IFUV into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the W-MSTN-IFUV, M-MSTN-IFUV or C-MSTN-IFUV recombinant protein is obtained through expression. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding IFUV by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-IFUV, M-MSTN-IFUV or C-MSTN-IFUV recombinant protein by using solid-phase synthesis technology; the chemical coupling includes chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with IFUV respectively to obtain W-MSTN-IFUV, M-MSTN-IFUV or C-MSTN-IFUV recombinant protein, or chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with IFUV respectively to obtain W-MSTN-IFUV, M-MSTN-IFUV or C-MSTN-IFUV recombinant protein.

[0199] Correspondingly, the present invention also provides:

[0200] I), the nucleic acid encoding the MSTN recombinant protein;

[0201] II), an expression unit containing the nucleic acid as described in I);

[0202] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0203] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0204] V), the culture product of the host cell as described in IV);

[0205] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0206] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the use of the above vaccine in increasing muscle, enhancing strength, or reducing body fat in certain diseases in humans or animals. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The administered immunization doses are 25 μg, 50 μg, 100 μg, and / or 200 μg. After immunization for 14 to 100 days, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat rate decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0207] In specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-Ferritin recombinant protein, an M-MSTN-Ferritin recombinant protein, or a C-MSTN-Ferritin recombinant protein. The Ferritin is from Helicobacter pylori and is a nanoparticle formed by the ferritin of Helicobacter pylori. The number of W-MSTN, M-MSTN, or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the n W-MSTN, M-MSTN, or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS. The flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the Ferritin is located at the N-terminus or C-terminus of W-MSTN, M-MSTN, or C-MSTN.

[0208] The amino acid sequence of the Ferritin includes:

[0209]

[0210] or a sequence obtained by substitution, deletion, addition, and / or replacement of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 18;

[0211] or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 18.

[0212] In the present invention, the preparation method of the Ferritin includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding Ferritin into an expression vector, and obtaining the Ferritin after expression by transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize Ferritin with an amino acid sequence as shown in SEQ ID NO: 18 by using solid-phase synthesis technology.

[0213] The present invention provides a preparation method of W-MSTN-Ferritin, M-MSTN-Ferritin or C-MSTN-Ferritin recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN and the nucleic acid encoding Ferritin into an expression vector respectively or in the form of a protein to obtain a recombinant expression vector, and obtain W-MSTN-Ferritin, M-MSTN-Ferritin or C-MSTN-Ferritin recombinant protein after expression by transferring the recombinant expression vector into a host. Among them, the fusion includes connecting the nucleic acid encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding Ferritin by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequence of W-MSTN-Ferritin, M-MSTN-Ferritin or C-MSTN-Ferritin recombinant protein by using solid-phase synthesis technology; the chemical coupling includes obtaining W-MSTN-Ferritin, M-MSTN-Ferritin or C-MSTN-Ferritin recombinant protein by chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with Ferritin respectively, or obtaining W-MSTN-Ferritin, M-MSTN-Ferritin or C-MSTN-Ferritin recombinant protein by chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with Ferritin respectively

[0214] Correspondingly, the present invention also provides:

[0215] I), nucleic acid encoding the MSTN recombinant protein;

[0216] II), an expression unit containing the nucleic acid as described in I);

[0217] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0218] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0219] V), a culture product of the host cell as described in IV);

[0220] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0221] Immunizing an animal with this vaccine can obtain the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength or reducing body fat in certain diseases in humans or animals. Correspondingly, the present invention also provides a method for testing the efficacy of a conjugate vaccine, including administering the vaccine as described above. The immunization dose administered is 25 μg, 50 μg, 100 μg and / or 200 μg. 14 to 100 days after immunization, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grasping force increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat rate decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0222] In the specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-Fc recombinant protein, an M-MSTN-Fc recombinant protein or a C-MSTN-Fc recombinant protein, and the Fc is derived from human and is the Fc-terminal protein of a human IgG antibody. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the Fc is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0223] The amino acid sequence of the Fc includes:

[0224]

[0225]

[0226] or a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 19;

[0227] or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 19.

[0228] In the present invention, the preparation method of the Fc includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: obtaining a recombinant expression vector by cloning the nucleic acid encoding Fc into an expression vector, and obtaining it by expression after transferring the recombinant expression vector into a host. The solid-phase synthesis is to synthesize the Fc with the amino acid sequence shown in SEQ ID NO: 19 by using solid-phase synthesis technology.

[0229] The present invention provides a method for preparing recombinant proteins of W-MSTN-Fc, M-MSTN-Fc or C-MSTN-Fc, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acids encoding W-MSTN, M-MSTN, C-MSTN and the nucleic acid encoding Fc into an expression vector separately or in a fused form to obtain a recombinant expression vector, and after transferring the recombinant expression vector into a host, the recombinant proteins of W-MSTN-Fc, M-MSTN-Fc or C-MSTN-Fc are obtained through expression. Among them, the fusion is to recombine the nucleic acids encoding W-MSTN, M-MSTN, C-MSTN with Fc through the nucleic acid encoding hinge and / or linker, and the amino acid sequence of the hinge includes: EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 20); the solid-phase synthesis is to synthesize the amino acid sequences of the recombinant proteins of W-MSTN-Fc, M-MSTN-Fc or C-MSTN-Fc by using solid-phase synthesis technology; the chemical coupling includes obtaining the recombinant proteins of W-MSTN-Fc, M-MSTN-Fc or C-MSTN-Fc by chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with Fc respectively, or obtaining the recombinant proteins of W-MSTN-Fc, M-MSTN-Fc or C-MSTN-Fc by chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with Fc respectively.

[0230] Correspondingly, the present invention also provides:

[0231] I), the nucleic acid encoding the MSTN recombinant protein;

[0232] II), an expression unit containing the nucleic acid as described in I);

[0233] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0234] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0235] V), the culture product of the host cell as described in IV);

[0236] The present invention also provides a vaccine containing the MSTN recombinant protein. The vaccine also includes an adjuvant. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0237] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength in humans or animals, or reducing body fat in certain diseases. Correspondingly, the present invention also provides a method for testing the efficacy of the conjugate vaccine, including administering the vaccine as described above. The administered immunization doses are 25 μg, 50 μg, 100 μg, and / or 200 μg. After immunization for 14 to 100 days, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat percentage decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0238] In specific embodiments of the present invention, the MSTN recombinant protein is a W-MSTN-MBP recombinant protein, an M-MSTN-MBP recombinant protein or a C-MSTN-MBP recombinant protein, and the MBP is maltose-binding protein. The number of W-MSTN, M-MSTN or C-MSTN is n, and n is an integer from 1 to 10, specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the n W-MSTN, M-MSTN or C-MSTN are connected by a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, and m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the MBP is located at the N-terminus or C-terminus of W-MSTN, M-MSTN or C-MSTN.

[0239] The amino acid sequence of the MBP includes:

[0240]

[0241] or a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 21;

[0242] or a sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 21.

[0243] In the present invention, the preparation method of the MBP includes solid-phase synthesis and / or biosynthesis; the biosynthesis is: a recombinant expression vector is obtained by cloning the nucleic acid encoding MBP into an expression vector, and after the recombinant expression vector is transferred into a host, it is obtained through expression. The solid-phase synthesis is to synthesize MBP with an amino acid sequence as shown in SEQ ID NO: 21 by using solid-phase synthesis technology.

[0244] The present invention provides a preparation method of W-MSTN-MBP, M-MSTN-MBP or C-MSTN-MBP recombinant protein, and the preparation method includes biosynthesis, solid-phase synthesis or chemical coupling. The biosynthesis is to clone the nucleic acids encoding W-MSTN, M-MSTN, C-MSTN and the nucleic acid encoding MBP into an expression vector respectively or in a fused form to obtain a recombinant expression vector, and after the recombinant expression vector is transferred into a host, W-MSTN-MBP, M-MSTN-MBP or C-MSTN-MBP recombinant protein is obtained through expression. Among them, the fusion includes connecting the nucleic acids encoding W-MSTN, M-MSTN or C-MSTN with the nucleic acid encoding MBP by using the nucleic acid encoding a linker, and the sequence of the linker is a flexible fragment composed of GS, and the flexible fragment includes (GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, specifically, it can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the solid-phase synthesis is to synthesize the amino acid sequences of W-MSTN-MBP, M-MSTN-MBP or C-MSTN-MBP recombinant protein by using solid-phase synthesis technology; the chemical coupling includes obtaining W-MSTN-MBP, M-MSTN-MBP or C-MSTN-MBP recombinant protein by chemically coupling the biosynthesized W-MSTN, M-MSTN or C-MSTN with MBP respectively, or obtaining W-MSTN-MBP, M-MSTN-MBP or C-MSTN-MBP recombinant protein by chemically coupling the solid-phase synthesized W-MSTN, M-MSTN or C-MSTN with MBP respectively

[0245] Correspondingly, the present invention also provides:

[0246] I), a nucleic acid encoding the MSTN recombinant protein;

[0247] II), an expression unit containing the nucleic acid as described in I);

[0248] III), a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II);

[0249] IV), a host cell transformed or transfected with the recombinant vector as described in III);

[0250] V), a culture product of the host cell as described in IV);

[0251] The present invention also provides a vaccine containing the MSTN recombinant protein. An adjuvant is also included in the vaccine. In the vaccine of the present invention, the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant, and the present invention does not limit this. In some embodiments, an aluminum adjuvant is used to prepare the vaccine of the MSTN recombinant protein. The concentration of GnRH-VLP in the vaccine is 800, 400, 200, 100 μg / mL, and preferably, the concentration is 200 μg / mL.

[0252] Immunizing animals with this vaccine can achieve the effects of increasing body weight, increasing muscle weight, enhancing skeletal muscle strength, and reducing body fat. Therefore, the present invention also provides the application of the above vaccine in increasing muscle, enhancing strength or reducing body fat in certain diseases in humans or animals. Correspondingly, the present invention also provides a method for testing the efficacy of a conjugate vaccine, including administering the vaccine as described above. The immunization dose administered is 25 μg, 50 μg, 100 μg and / or 200 μg. 14 to 100 days after immunization, the body weight or skeletal muscle increases by 5% to 40%, specifically 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, the grip strength increases by 3% to 5%, 5% to 10%, 10% to 15%, and the body fat rate decreases by 0.5% to 1.0%, 1.0% to 2.0%, 2.0% to 3.0%.

[0253] The present invention provides a method for preparing the MSTN recombinant protein, including culturing the host cell to obtain a culture product containing the MSTN recombinant protein.

[0254] The present invention provides a composition, including the MSTN recombinant protein, the MSTN antigen or any antigen that stimulates or inhibits the growth of animal skeletal muscle.

[0255] Furthermore, the composition further includes an acceptable adjuvant, and the adjuvant includes an aluminum salt adjuvant, a protein adjuvant, a nucleic acid adjuvant, a lipid-containing adjuvant, a mixed adjuvant or an aggregate structure adjuvant.

[0256] The present invention provides the application of the MSTN recombinant protein, the biomaterial or the composition in the preparation of a subunit vaccine.

[0257] The present invention provides an MSTN recombinant vaccine, which comprises the MSTN recombinant protein or the composition described in the present invention.

[0258] Furthermore, the MSTN recombinant protein may be a W-MSTN-AP205 recombinant protein, M-MSTN-AP205 recombinant protein, C-MSTN-AP205 recombinant protein, W-MSTN-Qβ recombinant protein, M-MSTN-Qβ recombinant protein, C-MSTN-Qβ recombinant protein, W-MSTN-MS2 recombinant protein, M-MSTN-MS2 recombinant protein, C-MSTN-MS2 recombinant protein, W-MSTN-T4 recombinant protein, M-MSTN-T4 recombinant protein, C-MSTN-T4 recombinant protein, W-MSTN-TMV recombinant protein, M-MSTN-TMV recombinant protein, C-MSTN-TMV recombinant protein, W-MSTN-CPMV recombinant protein, M-MSTN-CPMV recombinant protein, C-MSTN-CPMV recombinant protein, W-MSTN-CMV recombinant protein, M-MSTN-CMV recombinant protein, C-MSTN-CMV recombinant protein, W-MSTN-PapMV recombinant protein, M-MSTN-PapMV recombinant protein, C-MSTN-PapMV recombinant protein, W-MSTN-FHV recombinant protein, M-MSTN-FHV recombinant protein, C-MSTN-FHV recombinant protein, W-MSTN-HBsAg recombinant protein, M-MSTN-HBsAg recombinant protein, C-MSTN-HBsAg recombinant protein, W-MSTN-IFUV recombinant protein, M-MSTN-IFUV recombinant protein, C-MSTN-IFUV recombinant protein, W-MSTN-Ferritin recombinant protein, M-MSTN-Ferritin recombinant protein, C-MSTN-Ferritin recombinant protein, W-MSTN-Fc recombinant protein, M-MSTN-Fc recombinant protein, C-MSTN-Fc recombinant protein, W-MSTN-MBP recombinant protein, M-MSTN-MBP recombinant protein or C-MSTN-MBP recombinant protein, and the present invention does not make any limitation thereto.

[0259] In some embodiments of the present invention, the MSTN recombinant vaccine is an MSTN-conjugated virus-like particle, nanoparticle, or a vaccine formulated by fusion expression with a foreign fusion protein; the MSTN protein is the MSTN protein of a human or other animals, such as dogs, cats, pigs, chickens, etc., or a protein with more than 90% homology, and MSTN is a full-length protein, M-MSTN protein, or the C-terminal active region protein of MSTN; the VLP includes AP205, Qβ, MS2, T4, TMV, CPMV, CMV, PapMV, FHV, HBsAg, and / or IFUV, the nanoparticle includes Ferritin, etc., and the foreign fusion protein is such as Fc, MBP, etc. The present invention does not limit this.

[0260] The VLP, nanoparticle, and foreign fusion protein provided by the present invention have a variety of VLP, nanoparticle, and foreign fusion protein platforms, and do not limit the types of VLP, nanoparticle, and foreign fusion protein, increasing the types and quantities of MSTN conjugate protein vaccines.

[0261] The present invention also provides a preparation method of the MSTN recombinant vaccine, including mixing the MSTN recombinant protein with an adjuvant; or mixing the composition with a buffer solution.

[0262] The present invention provides a method for animals to increase body weight, increase skeletal muscle, increase muscle strength, and reduce body fat, including administering the MSTN recombinant vaccine of the present invention.

[0263] Animals that can be applied include birds and mammals, etc.

[0264] The present invention provides an MSTN recombinant protein, which has high antigen purity and good safety. When used for animal immunization, the prepared MSTN recombinant vaccine has no pathogenicity to animals such as mice, is easy to pass the safety evaluation, and at the same time, the vaccine has an efficient antigen presentation method, can effectively induce the immune system to produce an immune protection response, stimulate animal muscle growth, and has important application prospects in the prevention and treatment fields of related diseases such as muscle atrophy. Detailed implementation manners

[0265] The present invention provides an MSTN recombinant vaccine and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0266] The reagent consumables used in the present invention are all ordinary commercially available products and can be purchased on the market.

[0267] In the present invention, the nucleic acid can be DNA, RNA, cDNA or PNA. The form of DNA includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be linear or circular topologically. The nucleic acid can be, for example, part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic or chemical techniques. The form of RNA is mRNA obtained by gene transcription, etc. The gene sequence can adopt the wild-type sequence or can be codon-optimized, and the present invention does not limit this.

[0268] In the present invention, the expression unit includes an expression unit composed of the nucleic acid described in the present invention in single or multiple tandem forms and a promoter and a terminator, and the present invention does not limit this.

[0269] In the present invention, the recombinant expression vector refers to a nucleic acid vector, which is a recombinant DNA molecule that contains the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences or elements necessary for expression in bacteria include promoters, ribosome binding sites and possibly other sequences. The recombinant expression vector is selected according to the host. The expression vector described in the present invention can be circular or linear, and the present invention does not limit this.

[0270] Furthermore, the host described in the present invention includes bacteria, fungi, viruses or animals. The bacteria include Gram-positive bacteria and Gram-negative bacteria; the Gram-positive bacteria include, but are not limited to, Escherichia coli. The fungi include molds, yeasts, basidiomycetes; the yeasts include Saccharomyces cerevisiae, Saccharomyces cerevisiae, Pichia pastoris and Candida albicans, etc. The viruses include, but are not limited to, adenoviruses, adeno-associated viruses, lentiviruses, prions. The animals include humans, mice, rabbits, pigs, zebrafish, etc. The expression mode of the nucleic acid encoding the recombinant protein in the host can be integrative or free, and the present invention also does not limit this.

[0271] Taking prokaryotic hosts as an example, it can be Escherichia coli, or Bacillus subtilis, or Streptomyces or cyanobacteria, etc.; its plasmids can be pET series plasmids, pGEX series plasmids, pKBP series plasmids or pcDNA series plasmids.

[0272] The adjuvant described in the present invention is an immunomodulator or immunopotentiator, including aluminum salt adjuvants, protein adjuvants, nucleic acid adjuvants, lipid-containing adjuvants, mixed adjuvants or aggregate structure adjuvants.

[0273] In some embodiments of the present invention, the adjuvant is an aluminum hydroxide adjuvant.

[0274] In the present invention, the muscle atrophy-related diseases include muscle atrophy or loss caused by Becker muscular dystrophy, Duchenne muscular dystrophy, spinal muscular atrophy, pancreatic cancer, advanced kidney disease, etc.

[0275] The present invention will be further elaborated below in conjunction with examples:

[0276] Example 1 Preparation of W-MSTN protein or M-MSTN protein or C-terminal active region protein of MSTN

[0277] (1) Construction of recombinant plasmid: The nucleic acid sequence of human W-MSTN protein or M-MSTN protein or C-terminal active region protein of MSTN (C-MSTN), with 6 histidine (His) tags added at the C-terminus, was synthesized by BGI after codon optimization and cloned into the pcDNA3.4 vector.

[0278] (2) Preparation of HEK293F cells: An appropriate amount of HEK293F cells (about 1.5×10 6 cells / mL) were inoculated into a culture flask one day in advance. When the cell viability reached more than 90%, and the density was 23×10 6 cells / mL, transfection experiments could be carried out.

[0279] (3) Transient transfection and expression: Dilute 20 μg of pcDNA3.4 plasmid with 150 mM NaCl or serum-free medium to a total volume of 0.5 mL; dilute 60 μL of PEI transfection reagent (1 mg / mL) with 150 mM NaCl or serum-free medium to a total volume of 0.5 mL. Mix PEI with the pcDNA3.4 plasmid and let it stand for about 10 min to form the PEI-DNA complex. Add the transfection solution dropwise to the cell culture medium, gently shake the culture flask while dropping, and after mixing, return it to the shaker for continued culture. Loosen the bottle cap to meet the oxygen and CO 2 emission requirements for subsequent high-density cell growth, and prevent the pH value of the culture medium from being too low (the culture medium turns yellow) due to CO 2 accumulation, which affects cell growth.

[0280] (4) Protein purification: After culturing the transfected cells for 72 - 96 h, centrifuge to obtain the supernatant, filter it through a 0.22 μm filter membrane, and then perform affinity chromatography using a nickel column. After sample loading and equilibration, elute with a 0.8 mol / L imidazole eluent and collect the elution peak. This is the purified W-MSTN protein, M-MSTN protein, or the C-terminal active region protein of MSTN.

[0281] Example 2 Preparation of CMV VLP

[0282] (1) Construction of recombinant plasmid: After codon optimization of the nucleic acid sequence expressing CMV, it was synthesized by General Biosystems between the NdeI and XhoI restriction sites of the pET28a plasmid to obtain the recombinant plasmid CMV-pET28a;

[0283] (2) Transfection of recombinant plasmid into expression strain: The CMV-pET28a recombinant plasmid was transferred into the Escherichia coli BL21(DE3) expression strain to obtain the recombinant expression strain CMV-pET28a-BL21;

[0284] (3) Cultivation of recombinant bacteria and induction of target protein expression: The recombinant expression strain CMV-pET28a-BL21 was spread on a kanamycin-resistant plate containing 100 μg / mL and cultured overnight at 37 °C. Single colonies were picked and inoculated into 10 mL of LB medium containing 100 μg / mL kanamycin, and cultured with shaking at 37 °C and 200 rpm until the OD 600 of the bacterial solution reached 0.8 - 2.0. Then it was transferred to 500 mL of LB medium containing 100 μg / mL kanamycin and cultured with shaking at 37 °C and 200 rpm until the OD 600 of the bacterial solution reached about 0.8 - 1.5. An inducer of β-D-galactoside (IPTG) with a final concentration of 0.5 mmol / L was added, and induction expression was carried out overnight at 20 °C.

[0285] (4) Collection and disruption of bacteria: After centrifuging the bacterial solution at 8000 rpm, the bacteria were collected. The centrifuged bacteria were resuspended by adding a 0.01 mol / L PBS solution at a weight-to-volume ratio of 1:10. The parameters of the high-pressure homogenizer were set to 900 bar, and high-pressure disruption was performed 2 times at 2 - 8 °C.

[0286] (5) Removal of endotoxin: After disrupting the bacteria, the supernatant was obtained by centrifuging at 8000 rpm, and Triton X-114 was added to a final concentration of 1.5%, and stirred at 2 - 8 °C for 1 hour. After treatment, the sample temperature was restored to 30 °C and maintained for 40 minutes, and the precipitate was removed by centrifugation, and the supernatant was collected. The above method was repeated 2 times.

[0287] (6) Protein purification: After the endotoxin-removed protein solution is concentrated and buffer-exchanged using a 100 KD membrane package, it is chromatographically purified using S300 molecular sieve chromatography packing material, and the target protein peak is collected, which is the purified CMV virus-like particle protein.

[0288] Example 3 Preparation of PapMV VLPs

[0289] (1) Construction of recombinant plasmid: After the nucleic acid sequence expressing PapMV is codon-optimized, it is synthesized by BGI between the NdeI and XhoI restriction sites of the pET28a plasmid to obtain the recombinant plasmid PapMV-pET28a.

[0290] (2) Transfection of recombinant plasmid into expression strain: The PapMV-pET28a recombinant plasmid is transferred into the Escherichia coli BL21(DE3) expression strain to obtain the recombinant expression strain PapMV-pET28a-BL21.

[0291] (3) Cultivation of recombinant bacteria and induction of target protein expression: The recombinant expression strain PapMV-pET28a-BL21 is spread on a kanamycin-resistant plate containing 100 μg / mL and cultured overnight at 37°C. A single colony is picked and inoculated into 10 mL of LB medium containing 100 μg / mL kanamycin, and shaken at 37°C and 200 rpm until the OD 600 of the bacterial solution reaches 0.8 - 2.0. It is transferred to 500 mL of LB medium containing 100 μg / mL kanamycin and shaken at 37°C and 200 rpm until the OD 600 of the bacterial solution reaches about 0.8 - 1.5. An inducer of β-D-galactoside (IPTG) with a final concentration of 0.5 mmol / L is added, and induced expression is carried out overnight at 20°C.

[0292] (4) Collection and disruption of bacterial cells: After the bacterial solution is centrifuged at 8000 rpm, the bacterial cells are collected. The centrifuged bacterial cells are resuspended in 0.01 mol / L PBS solution according to the weight-to-volume ratio of 1:10, and the parameters of the high-pressure homogenizer are set to 900 bar, and high-pressure disruption is carried out twice at 2 - 8°C.

[0293] (5) Endotoxin removal: After the bacterial cells are disrupted, the supernatant is taken by centrifugation at 8000 rpm, and Triton X-114 is added to a final concentration of 1.5%, and stirred at 2 - 8°C for 1 hour. After treatment, the sample temperature is restored to 30°C and maintained for 40 minutes, and the precipitate is removed by centrifugation, and the supernatant is collected. The above method is repeated twice.

[0294] (6) Protein purification: After the endotoxin-removed protein solution is concentrated and buffer-exchanged using a 100 KD membrane package, it is chromatographically purified using S300 molecular sieve chromatography packing material, and the target protein peak is collected, which is the purified PapMV virus-like particle protein.

[0295] Preparation of Example 4 AP205 VLP

[0296] (1) Construction of recombinant plasmid: After codon optimization of the nucleic acid sequence expressing AP205, it was synthesized by BGI between the NdeI and XhoI restriction sites of the pET28a plasmid to obtain the recombinant plasmid AP205-pET28a;

[0297] (2) Transfection of recombinant plasmid into expression strain: The AP205-pET28a recombinant plasmid was transferred into the Escherichia coli BL21(DE3) expression strain to obtain the recombinant expression strain AP205-pET28a-BL21;

[0298] (3) Cultivation of recombinant bacteria and induction of expression of target protein: The recombinant expression strain AP205-pET28a-BL21 was spread on a kanamycin-resistant plate containing 100 μg / mL and cultured overnight at 37 °C. Single colonies were picked and inoculated into 10 mL of LB medium containing 100 μg / mL kanamycin, and cultured with shaking at 200 rpm at 37 °C until the OD 600 of the bacterial solution was 0.8 - 2.0. It was transferred to 500 mL of LB medium containing 100 μg / mL kanamycin and cultured with shaking at 200 rpm at 37 °C until the OD 600 of the bacterial solution was about 0.8 - 1.5. An inducer of β-D-galactoside (IPTG) with a final concentration of 0.5 mmol / L was added, and induced expression was carried out overnight at 20 °C.

[0299] (4) Collection and disruption of bacteria: After centrifugation of the bacterial solution at 8000 rpm, the bacteria were collected. The centrifuged bacteria were resuspended by adding 0.01 mol / L PBS solution according to the weight-to-volume ratio of 1:10. The parameters of the high-pressure homogenizer were set to 900 bar, and high-pressure disruption was carried out 2 times at 2 - 8 °C.

[0300] (5) Removal of endotoxin: After disruption of the bacteria, the supernatant was taken by centrifugation at 8000 rpm, and Triton X-114 was added to a final concentration of 1.5%, and stirred at 2 - 8 °C for 1 hour. After treatment, the sample temperature was restored to 30 °C and maintained for 40 minutes, and the precipitate was removed by centrifugation, and the supernatant was collected. The above method was repeated 2 times.

[0301] (6) Protein purification: The protein solution after endotoxin removal was concentrated and buffer exchanged through a 100 KD membrane package, and then chromatographically purified using S300 molecular sieve chromatography packing material. The target protein peak was collected, which was the purified AP205 virus-like particle protein.

[0302] Preparation of Example 5 Qβ VLP

[0303] (1) Construction of recombinant plasmid: After codon optimization of the nucleic acid sequence expressing Qβ, it was synthesized by BGI between the NdeI and XhoI restriction sites of the pET28a plasmid to obtain the recombinant plasmid Qβ-pET28a;

[0304] (2) Transfection of recombinant plasmid into expression strain: The Qβ-pET28a recombinant plasmid was transferred into the Escherichia coli BL21(DE3) expression strain to obtain the recombinant expression strain Qβ-pET28a-BL21;

[0305] (3) Cultivation of recombinant bacteria and induction expression of target protein: The recombinant expression strain Qβ-pET28a-BL21 was spread on a kanamycin-resistant plate containing 100 μg / mL and cultured overnight at 37 °C. Single colonies were picked and inoculated into 10 mL of LB medium containing 100 μg / mL kanamycin, and cultured with shaking at 37 °C and 200 rpm until the OD 600 of the bacterial solution reached 0.8 - 2.0. Then it was transferred to 500 mL of LB medium containing 100 μg / mL kanamycin and cultured with shaking at 37 °C and 200 rpm until the OD 600 of the bacterial solution reached about 0.8 - 1.5. An inducer of β-D-galactoside (IPTG) with a final concentration of 0.5 mmol / L was added, and induced expression was carried out overnight at 20 °C.

[0306] (4) Collection and disruption of bacteria: After centrifugation of the bacterial solution at 8000 rpm, the bacteria were collected. The centrifuged bacteria were resuspended by adding 0.01 mol / L PBS solution according to the weight-to-volume ratio of 1:10. The parameters of the high-pressure homogenizer were set to 900 bar, and high-pressure disruption was carried out 2 times at 2 - 8 °C.

[0307] (5) Removal of endotoxin: After disruption of the bacteria, the supernatant was taken by centrifugation at 8000 rpm, and Triton X-114 was added to a final concentration of 1.5%, and stirred at 2 - 8 °C for 1 hour. After treatment, the sample temperature was restored to 30 °C and maintained for 40 minutes, and the precipitate was removed by centrifugation, and the supernatant was collected. The above method was repeated 2 times.

[0308] (6) Protein purification: After the endotoxin-removed protein solution was concentrated and buffer exchanged through a 100KD membrane package, chromatography purification was carried out using S300 molecular sieve chromatography packing, and the target protein peak was collected, which was the purified Qβ virus-like particle protein.

[0309] Example 6 Preparation of MS2 VLP

[0310] Escherichia coli MS2 phage belongs to the positive-sense single-stranded RNA spherical virus, with a full-length genome of 3659 bp, encoding four protein molecules, namely maturase protein, capsid protein, replicase protein, and lysis protein. Research has found that in vitro, the genes of the maturase protein and capsid protein of MS2 phage, as well as the gene of the 5' non-coding sequence containing gene regulatory elements, are cloned into an expression vector, and the induced-expressed proteins can self-assemble into mature virus-like particles (VLPs). Moreover, inserting several genes at specific sites in the capsid protein gene can be expressed as VLPs with exogenous amino acid display.

[0311] (1) Construction of recombinant plasmid: The gene sequences of maturase protein and capsid protein were commissioned to a gene company to be synthesized into the pET28a plasmid vector, obtaining the recombinant plasmid MS2-pET28a.

[0312] (2) Transfer of recombinant plasmid into expression strain: The recombinant plasmid MS2-pET28a was transferred into the Escherichia coli expression strain BL21(DE3) to obtain the MS2-pET28a-BL21 recombinant expression strain.

[0313] (3) Preparation of recombinant plasmid MS2-pET28a: 5 μL of glycerol bacteria containing the plasmid was pipetted and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin), and cultured with shaking at 37 °C for 14 - 16 hours. After culturing, 1 mL of the bacterial solution was sent for sequencing. The remaining bacterial solution was used to extract the plasmid with a plasmid mini-prep kit, and the nucleic acid concentration was detected with a Nanodrop2000 nucleic acid detector.

[0314] (4) Bacterial culture and MS2 VLP expression: The BL21(DE3) expression strain was transformed, centrifuged and concentrated after shaking the bacteria, spread on a kanamycin-resistant plate, and cultured overnight at 37 °C. The colonies were scraped and inoculated into 10 mL of LB medium containing kanamycin, and shaken at 37 °C and 200 rpm until the OD 600 value was about 0.8. It was transferred to 500 mL of LB medium containing kanamycin, and shaken at 37 °C and 180 rpm until the OD 600 value was about 0.8. IPTG inducer was added, and expression was carried out overnight at 30 °C. On the third day, the bacteria were collected by centrifugation at 4500 rpm for 15 min. The bacteria were ultrasonically disrupted for 30 min. After the ultrasonication, the supernatant was collected by centrifugation at 9500 rpm for 20 min at 4 °C.

[0315] (5) Endotoxin removal: After the bacteria were disrupted, the supernatant was taken by centrifugation at 8000 rpm, and Triton X-114 was added to a final concentration of 1.5%, and stirred at 2 - 8 °C for 1 hour. After treatment, the sample temperature was restored to 30 °C and maintained for 40 minutes, and the precipitate was removed by centrifugation, and the supernatant was collected. The above method was repeated 2 times.

[0316] (6) Protein purification: After the protein solution with endotoxin removed is concentrated and buffer-exchanged through a 100 KD membrane package, it is chromatographically purified using S300 molecular sieve chromatography packing material, and the target protein peak is collected, which is the purified Qβ virus-like particle protein.

[0317] Example 7 Preparation of T4 VLP

[0318] The capsid of bacteriophage T4 contains two non-essential outer proteins, Soc and Hoc, which allow for the high-density arrangement of antigenic epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Bacteriophage T4 VLP has high immunogenicity, does not require adjuvants, and provides complete protection against bacterial and viral pathogens. The T4 capsid gene sequence or the Soc or Hoc gene sequence is cloned into a prokaryotic expression vector to obtain a recombinant expression vector, and T4 VLP can be obtained through the expression and purification of the Escherichia coli expression system.

[0319] (1) Construction of recombinant plasmid: The T4 Soc or Hoc gene sequence is entrusted to a gene company to be synthesized into the pET28a plasmid vector to obtain the recombinant plasmid T4-pET28a.

[0320] (2) Transformation of recombinant plasmid into expression strain: The recombinant plasmid T4-pET28a is transformed into the Escherichia coli expression strain BL21(DE3) to obtain the T4-pET28a-BL21 recombinant expression strain.

[0321] (3) Preparation of recombinant plasmid T4-pET28a: 5 μl of glycerol bacteria containing the plasmid is pipetted and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin), and cultured with shaking at 37 °C for 14 - 16 hours. After culturing, 1 mL of the bacterial solution is sent for sequencing. The remaining bacterial solution is used to extract the plasmid with a plasmid miniprep kit, and the nucleic acid concentration is detected with a Nanodrop2000 nucleic acid detector.

[0322] (4) Bacterial culture and T4 VLP expression: The BL21(DE3) expression strain is transformed, centrifuged and concentrated after shaking the bacteria, spread on a kanamycin-resistant plate, and cultured overnight at 37 °C. The colonies are scraped off and inoculated into 10 mL of LB medium containing kanamycin, and shaken at 37 °C and 200 rpm until the OD 600 value of the bacterial solution is about 0.8. It is transferred to 500 mL of LB medium containing kanamycin, and shaken at 37 °C and 180 rpm until the OD 600 value of the bacterial solution is about 0.8. IPTG inducer is added, and expression is carried out overnight at 30 °C, and the bacterial solution is harvested.

[0323] (5) Bacterial cell collection and disruption: After centrifuging the bacterial solution at 8000 rpm, the bacterial cells were collected. The centrifuged bacterial cells were taken, and a 0.01 mol / L PBS solution was added according to the weight-to-volume ratio of 1:10, resuspended, and the parameters of the high-pressure homogenizer were set to 900 bar, and high-pressure disruption was carried out twice at 2-8 °C.

[0324] (6) Endotoxin removal: After disrupting the bacterial cells, the supernatant was taken by centrifuging at 8000 rpm, and Triton X-114 was added to a final concentration of 1.5%, and stirred at 2-8 °C for 1 hour. After treatment, the sample temperature was restored to 30 °C and maintained for 40 minutes, and the precipitate was removed by centrifugation, and the supernatant was collected. The above method was repeated 2 times.

[0325] (7) Protein purification: After the endotoxin-removed protein solution was concentrated and exchanged with a 100KD membrane package, chromatography purification was carried out using S300 molecular sieve chromatography packing, and the target protein peak was collected, which was the purified T4 virus-like particle protein.

[0326] Example 8 Preparation of TMV VLPs

[0327] Tobacco mosaic virus (TMV) is a plant virus that cannot infect animal cells, but has been shown to interact with mammalian dendritic cells and stimulate them, triggering an immune response.

[0328] (1) Construction of recombinant plasmid: After optimizing the codons of the TMV gene, it was synthesized by BGI into the pKBP121 expression vector to obtain the recombinant plasmid TMV-pKBP121.

[0329] (2) Transfer of recombinant plasmid into plant expression system: The recombinant plasmid TMV-pKBP121 was transiently transfected into Nicotiana benthamiana (Nb).

[0330] (3) Purification of TMV-pKBP121: The transfected plants were harvested, the soluble protein fraction was isolated, and TMV VLPs were purified by a combination of protein A affinity and anion exchange chromatography.

[0331] Example 9 Preparation of CPMV VLPs

[0332] (1) Construction of recombinant plasmid: After optimizing the nucleic acid sequence expressing CPMV, it was synthesized by BGI between the NdeI restriction site and the XhoI restriction site of the pET28a plasmid to obtain the recombinant plasmid CPMV-pET28a;

[0333] (2) Transfection of the recombinant plasmid into the expression strain: The CPMV-pET28a recombinant plasmid was transferred into the Escherichia coli BL21(DE3) expression strain to obtain the recombinant expression strain CPMV-pET28a-BL21;

[0334] (3) Cultivation of the recombinant bacterium and induction of expression of the target protein: The recombinant expression strain CPMV-pET28a-BL21 was spread on a kanamycin-resistant plate containing 100 μg / mL and cultured overnight at 37 °C. Single colonies were picked and inoculated into 10 mL of LB medium containing 100 μg / mL kanamycin, and shaken at 37 °C and 200 rpm until the OD 600 of the bacterial solution was 0.8 - 2.0. It was transferred to 500 mL of LB medium containing 100 μg / mL kanamycin and shaken at 37 °C and 200 rpm until the OD 600 of the bacterial solution was about 0.8 - 1.5. An inducer of β-D-galactoside (IPTG) with a final concentration of 0.5 mmol / L was added, and induction expression was carried out overnight at 20 °C.

[0335] (4) Collection and disruption of the bacterial cells: After centrifugation of the bacterial solution at 8000 rpm, the bacterial cells were collected. The centrifuged bacterial cells were resuspended by adding PBS solution at 0.01 mol / L according to the weight-to-volume ratio of 1:10. The parameters of the high-pressure homogenizer were set at 900 bar, and high-pressure disruption was carried out 2 times at 2 - 8 °C.

[0336] (5) Removal of endotoxin: After disruption of the bacterial cells, the supernatant was taken by centrifugation at 8000 rpm, and Triton X-114 was added to a final concentration of 1.5%, and stirred at 2 - 8 °C for 1 hour. After treatment, the sample temperature was restored to 30 °C and maintained for 40 minutes, and the precipitate was removed by centrifugation, and the supernatant was collected. The above method was repeated 2 times.

[0337] (6) Protein purification: After the protein solution with endotoxin removed was concentrated and exchanged with a 100 KD membrane package, chromatography purification was carried out using S300 molecular sieve chromatography packing, and the target protein peak was collected, which was the purified CPMV virus-like particle protein.

[0338] Example 10 Preparation of FHV VLP

[0339] Flock House Virus (FHV), an unenveloped insect RNA virus, has an icosahedral capsid composed of 180 identical coat protein subunits. Due to its genetic simplicity, special yield, and high physicochemical stability, and since there are no known safety issues in the application of FHV in animals and humans, this virus is very suitable for various biotechnological applications.

[0340] (1) Construction of recombinant plasmid: After optimizing the codons of the FHV capsid protein gene sequence, it was entrusted to a gene company for synthesis into the pBacPAK9 plasmid vector to obtain the recombinant plasmid FHV-pBacPAK9;

[0341] (2) Transfer of recombinant plasmid into insect cells: The recombinant plasmid FHV-pBacPAK9 was transiently transfected into insect cells, and the supernatant was collected. The supernatant was then used to infect SF9 insect cells again;

[0342] (3) Purification of FHV VLPs: When the viability of SF9 cells decreased to less than 30%, the supernatant was collected by centrifugation. After filtration through a 0.22 μm filter membrane, it was subjected to Q column chromatography. Then, it was concentrated and buffer exchanged using a 100 KD membrane package, and further purified by chromatography using S300 molecular sieve chromatography packing. The target protein peak was collected, which was the purified PapMV virus-like particle protein.

[0343] Example 11 Preparation of HBsAg VLPs

[0344] Hepatitis B vaccines are based on the major hepatitis B surface antigen (HBsAg), and this protein can assemble into so-called virus-like particles (VLPs).

[0345] (1) Construction of recombinant plasmid: After optimizing the codons of the HBsAg gene, it was entrusted to a gene company for synthesis into the yeast expression vector pPIC9K to obtain the recombinant plasmid HBs-pPIC9K;

[0346] (2) Linearization of recombinant plasmid: The recombinant plasmid was digested with a single enzyme and then linearized;

[0347] (3) Electroporation of yeast GS115 strain: The linearized recombinant plasmid HBs-pPIC9K was electroporated into the yeast GS115 strain, and the cells were inoculated on an MD plate for screening;

[0348] (4) Protein expression: Monoclonal colonies were selected from the MD plate and all colonies on the YPD plate containing 5.0 mg / mL G418. They were cultured in a 100 mL Erlenmeyer flask containing 20 mL of BMGY medium at 28 °C and 200 rpm. When the OD of the culture 600 reached 20 - 24, the cells were resuspended in a 100 mL Erlenmeyer flask containing 20 mL of BMGY medium, and pure methanol was added to the culture flask every 24 h at a concentration of 1 - 5% for induced expression. After 96 h of induction, the culture was centrifuged at 10000 g to collect the culture.

[0349] (5) Collection and disruption of bacterial cells: The centrifuged bacterial cells were taken, and a 0.01 mol / L PBS solution was added according to the weight-to-volume ratio of 1:10, and they were resuspended. The parameters of the high-pressure homogenizer were set to 1300 bar, and the cells were disrupted under high pressure at 2 - 8 °C for 3 times.

[0350] (6) Endotoxin removal: After cell disruption, the supernatant was collected by centrifugation at 8000 rpm and preliminarily purified using an ion exchange chromatography column. Then, chromatography purification was performed using S300 molecular sieve chromatography packing, and the target protein peak was collected, which was the purified HBsAg VLP.

[0351] Example 12 Preparation of IFUV VLP

[0352] (1) Construction of recombinant plasmid: After codon optimization of the HA gene and M1 gene of influenza virus, they were synthesized by a gene synthesis company onto pFastBac I to obtain the donor plasmid pFastBac-M1-HA with tandem expression of the two genes.

[0353] (2) Construction and identification of recombinant Bacmid: 2 μl of the obtained donor plasmid pFastBac-M1-HA with tandem expression of the two genes was transfected into DH10Bac competent cells. In SOC medium, they were cultured at 37 °C and 200 rpm for 4 h. 100 μl of the bacterial solution was spread on a plate containing IPTG / X-gal / carbenicillin / tetracycline / gentamicin triple-antibody, and cultured at 37 °C for more than 48 h. When blue and white colonies were obvious, a single white colony was picked and cultured overnight in 5 mL of liquid LB medium with carbenicillin / tetracycline / gentamicin triple-antibody. After the PCR product was identified as correct, the recombinant bacmid was extracted.

[0354] (3) Transfect the recombinant bacmid into SF9 insect cells. After culturing for 72 h, collect the supernatant virus solution.

[0355] (4) Preparation and purification of VLP: The collected virus solution was inoculated into SF9 cells cultured in suspension. They were cultured at 27 °C on a shaker at 110 rpm until the cell viability was below 20%. The virus solution was harvested. After centrifugation to collect the supernatant, it was filtered through a 0.22 μm filter membrane, then subjected to Q column chromatography, concentrated and buffer exchanged using a 100 KD membrane package, and then chromatography purification was performed using S300 molecular sieve chromatography packing, and the target protein peak was collected, which was the purified IFUV VLP.

[0356] Example 13 Preparation of Ferritin nanoparticles

[0357] (1) Construction of recombinant plasmid: After codon optimization of the nucleic acid sequence expressing Ferritin, it was synthesized by a gene synthesis company between the NdeI restriction site and XhoI restriction site of the pET28a plasmid to obtain the corresponding recombinant plasmid Ferritin-pET28a;

[0358] (2) Transfer the recombinant plasmid into the expression strain: Transfer the above recombinant plasmid into the Escherichia coli BL21(DE3) expression strain to obtain the recombinant expression strain;

[0359] (3) Bacterial culture and virus-like particle purification: Culture the recombinant expression strain, and induce expression with IPTG to obtain the corresponding virus-like particles.

[0360] (4) Endotoxin removal: After disrupting the bacteria, centrifuge at 8000 rpm and take the supernatant. Add Triton X-114 to a final concentration of 1.5%, and stir at 2-8 °C for 1 hour. After treatment, restore the sample temperature to 30 °C and maintain it for 40 minutes, centrifuge to remove the precipitate, and collect the supernatant. Repeat the treatment 2 times according to the above method.

[0361] (5) Protein purification: After concentrating and exchanging the endotoxin-free protein solution through a 100KD membrane package, perform chromatography purification using S300 molecular sieve chromatography packing, and collect the target protein peak, which is the purified virus-like particle protein.

[0362] Example 14 Fusion Expression of W-MSTN Protein or M-MSTN Protein or C-terminal Active Region Protein of MSTN with Fc

[0363] (1) Construction of recombinant plasmid: Connect human W-MSTN protein or M-MSTN protein or C-terminal active region protein of MSTN to the Fc end through hinge (amino acid sequence: EPKSCDKTHTCPPCPAPELLGG), and then optimize the codons of the connected protein gene, and clone it into the pcDNA3.4 vector by a gene synthesis company.

[0364] (2) Preparation of HEK293F cells: Inoculate an appropriate amount of HEK293F cells (about 1.5×10 6 cells / mL) into a culture flask one day in advance. When the cell viability reaches more than 90% and the density is 2-3×10 6 cells / mL, transfection experiments can be carried out.

[0365] (3) Transient transfection and expression: Dilute 20 μg of pcDNA3.4 plasmid with 150 mM NaCl or serum-free medium to a total volume of 0.5 mL; dilute 60 μL of PEI transfection reagent (1 mg / mL) with 150 mM NaCl or serum-free medium to a total volume of 0.5 mL, mix PEI with the pcDNA3.4 plasmid, and let it stand for about 10 min to form the PEI-DNA complex. Add the transfection solution dropwise to the cell culture medium, gently shake the culture flask while dropping, and after mixing, return it to the shaker for continued culture. Loosen the bottle cap to meet the oxygen demand and CO2 emission requirements for subsequent high-density cell growth, and prevent the pH value of the culture medium from being too low (the culture medium turns yellow) due to CO 2 accumulation, which affects cell growth.

[0366] (4) Protein purification: After culturing the transfected cells for 72 - 96 h, centrifuge to obtain the supernatant, and perform affinity chromatography using Protein A affinity chromatography. After sample loading and equilibration, elute and collect the elution peak. This is the purified W-MSTN or M-MSTN or the C-terminal active region fusion Fc protein of MSTN.

[0367] Example 15 Expression of W-MSTN protein or M-MSTN protein or the C-terminal active region protein of MSTN fused with MBP

[0368] (1) Construction of recombinant plasmid: Connect the human W-MSTN protein or M-MSTN protein or the C-terminal active region protein of MSTN through GS or GGS or GGGS or GGGGS or GSG)m, where m is an integer between 1 and 10, to the N-terminal of MBP. Then, after codon optimization of the ligated protein gene, it is synthesized by a gene synthesis company and cloned into the pcDNA3.4 vector.

[0369] (2) Preparation of HEK293F cells: Inoculate an appropriate amount of HEK293F cells (about 1.5×10 6 cell / mL) into a culture flask one day in advance. When the cell viability reaches over 90%, with a density of (2 - 3)×10 6 cell / mL, transfection experiments can be carried out.

[0370] (3) Transient transfection and expression: Dilute 20 μg of pcDNA3.4 plasmid with 150 mM NaCl or serum-free medium to a total volume of 0.5 mL; dilute 60 μL of PEI transfection reagent (1 mg / mL) with 150 mM NaCl or serum-free medium to a total volume of 0.5 mL. Mix PEI with the pcDNA3.4 plasmid and let it stand for about 10 min to form the PEI-DNA complex. Add the transfection solution dropwise into the cell culture medium, gently shake the culture flask while adding, and after mixing, return it to the shaker for continued culture. Loosen the bottle cap to meet the oxygen and CO 2 emission requirements for subsequent high-density cell growth, and prevent the pH value of the culture medium from being too low (the culture medium turns yellow) due to CO 2 accumulation, which affects cell growth.

[0371] (4) Protein purification: After culturing the transfected cells for 72 - 96 h, centrifuge to obtain the supernatant, and perform affinity chromatography using Protein A affinity chromatography. After sample loading and equilibration, elute and collect the elution peak. This is the purified W-MSTN or M-MSTN or the C-terminal active region fusion MBP protein.

[0372] Example 16 Coupling of W-MSTN protein, M-MSTN protein or C-terminal active region protein of MSTN with AP205 VLP, QβVLP, MS2 VLP, T4 VLP, TMV VLP, CPMV VLP, CMV VLP, PapMV VLP, FHV VLP, EILV VLP, HBsAgVLP, Ferritin nanoparticles

[0373] (1) Coupling: Use chemical reagents SMPH, i.e., succinimidyl 6-((β-maleimidopropionamido)hexanoate), and TCEP.HCL, i.e., tris(2-carboxyethyl)phosphine hydrochloride, for chemical coupling between proteins. Among them, the molar concentration ratio of SMPH to VLP or Ferritin nanoparticles is mixed at 5-10:1; the molar concentration ratio of TCEP.HCL to W-MSTN protein, M-MSTN protein or C-terminal active region protein of MSTN is mixed at 5-10:1; they are respectively mixed evenly in a thermostat at 25°C and 400 rpm for 30 min for coupling. Then, the two kinds of coupled protein solutions are mixed according to a molar concentration ratio of 1:2, and mixed evenly in a thermostat at 25°C and 400 rpm for 30 min. The VLP of the coupled protein is thus obtained.

[0374] (2) Nickel ion affinity chromatography purification: Pass the coupled protein solution through a nickel column. After sample loading and equilibration, elute with 0.8 mol / L imidazole eluent and collect the elution peak.

[0375] (3) Molecular sieve chromatography: Use S300 molecular sieve chromatography packing for chromatography purification, and collect the target protein peak, which is the purified VLP coupled with W-MSTN protein, M-MSTN protein or C-terminal active region protein of MSTN, or the nanoparticles of W-MSTN protein, M-MSTN protein or C-terminal active region protein of MSTN.

[0376] Example 17 Preparation of MSTN combined protein vaccine and detection of body weight, muscle and muscle contractility of ordinary mice

[0377] (1) Vaccine preparation and immunization: Take the purified VLP or nanoparticles coupled with W-MSTN protein, M-MSTN protein or C-terminal active region protein of MSTN, or the recombinant protein fused with Fc, or the recombinant protein fused with MBP, and mix them with aluminum hydroxide adjuvant according to a volume ratio of 1:1 to prepare the vaccine. The virus-like particle concentration is the protein concentration of 200 μg / mL. Take 5-week-old male C57BL / 6J mice with similar body weights, 5 in each group, for immunization, 0.5 mL / mouse. The immunization program is to immunize once at 0 day and 14 days respectively. At the same time, set up a control group of 5 mice injected with the same dose of adjuvant and a control group of 5 mice injected with the same dose of normal saline.

[0378] (2) Detection: The body weights of these mice were measured on days 0, 14, 28, and 42 respectively; on day 42 after immunization, a quantitative nuclear magnetic resonance instrument was used to measure the body fat of the mice, so as to calculate the weight excluding body fat, and indirectly analyze the overall muscle growth of the mice; a grip strength meter was used to measure the grip strength of the mice, and the grip strength of each group of mice was compared to test the muscle strength; after the mice were sacrificed, the weight of the leg muscles was measured.

[0379] Detection of the weight loss effect of the MSTN combined protein vaccine on obese mice in Example 18

[0380] (1) Immunization: Obese C57BL / 6J mice of 5 weeks old with similar body weights, which were obese mice caused by gene mutation, 5 mice in each group, were immunized, 0.5 mL per mouse, and the immunization procedure was to immunize once on days 0 and 14 respectively. At the same time, 5 mice in the control group injected with the same dose of adjuvant and 5 mice in the control group injected with the same dose of normal saline were set up.

[0381] (2) Detection of body weight, body fat, and leg muscle weight: The body weights of these mice were measured on days 0, 14, 28, and 42 after immunization respectively. A quantitative nuclear magnetic resonance instrument was used to measure the body fat of the mice, so as to calculate the weight excluding body fat, and indirectly analyze the overall muscle growth of the mice. The mice were sacrificed, the skin was stripped, photographed, the abdominal cavity was opened, and photographed to visually observe the body fat and muscle morphology and quantity of the mice. The hind leg muscles of the mice were stripped and weighed to compare the weight of the leg muscles.

[0382] (3) Serological detection: When the mice were sacrificed, blood was collected to measure the MSTN antibody level. Blood routine (blood glucose, blood lipid, Leptin, cholesterol) and insulin level were detected.

[0383] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. MSTN recombinant protein, characterized in that, it comprises at least one of virus-like particle protein, nanoparticle protein, Fc or MBP and MSTN; the virus-like particle protein comprises AP205, Qβ, MS2, T4, TMV, CPMV, CMV, PapMV, FHV, HBsAg and / or IFUV; the nanoparticle protein comprises Ferritin.

2. The MSTN recombinant protein according to claim 1, characterized in that, the MSTN has the amino acid sequence shown as follows: (1) the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; or (2) a sequence with one or more amino acids substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown in (1); or (3) a sequence with a homology of more than 90% with the amino acid sequence shown in (1); the AP205 is from the capsid of AP205 phage; the Qβ is from the capsid of Qβ phage; the MS2 is from the capsid of MS2 phage; the T4 is from the capsid of T4 phage; the TMV is from tobacco mosaic virus; the CPMV is from cowpea mosaic virus; the CMV is from cucumber mosaic virus; the PapMV is from papaya mosaic virus; the FHV is from barn virus; the HBsAg is from hepatitis B surface antigen; the IFUV is from influenza matrix M1 protein; the Ferritin is from Helicobacter pylori; the Fc protein is the Fc-terminal protein of IgG antibody; the MBP is maltose-binding protein.

3. Biological material, characterized in that, it comprises any of the following: I) nucleic acid encoding the MSTN recombinant protein according to claim 1 or 2; II) an expression unit containing the nucleic acid as described in I); III) a recombinant vector containing the nucleic acid as described in I) or the expression unit as described in II); IV) a host cell transformed or transfected with the recombinant vector as described in III); V) a culture product of the host cell as described in IV).

4. Preparation method of the MSTN recombinant protein according to claim 1 or 2, characterized in that, it comprises culturing the host cell described in claim 3 to obtain a culture product containing the MSTN recombinant protein according to claim 1 or 2.

5. Composition, characterized in that, it comprises the MSTN recombinant protein according to claim 1 or 2, MSTN antigen or any antigen that stimulates or inhibits animal muscle growth.

6. The composition according to claim 5, characterized in that, it further comprises an acceptable adjuvant; the adjuvant comprises aluminum salt adjuvant, protein adjuvant, nucleic acid adjuvant, lipid-containing adjuvant, mixed adjuvant or aggregate structure adjuvant.

7. Application of the MSTN recombinant protein according to claim 1 or 2, the biological material according to claim 3 or the composition according to claim 5 or 6 in the preparation of a recombinant vaccine and / or a drug for preventing and treating muscle atrophy-related diseases.

8. MSTN recombinant vaccine, characterized in that, The raw materials include the MSTN recombinant protein described in claim 1 or 2 or the composition described in claim 5 or 6.

9. The method for preparing the MSTN recombinant vaccine according to claim 8, characterized in that it includes mixing the MSTN recombinant protein described in claim 1 or 2 with an adjuvant; or mixing the composition described in claim 5 or 6 with a buffer.

10. The method for stimulating animal muscle growth, characterized in that it includes administering the MSTN recombinant vaccine according to claim 8.

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