Castration MS2 virus-like particle subunit vaccine and preparation method thereof

Abstract

The invention discloses a GNRH-I-MS2 fusion protein, a castrated MS2 virus-like particle subunit vaccine and a preparation method of the castrated MS2 virus-like particle subunit vaccine.

Description

Castration-resistant MS2 virus-like particle subunit vaccine and preparation method thereof

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on March 21, 2023, with application number 202310317333.6 and invention name “GnRH-VLP recombinant castration vaccine and its preparation method”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biotechnology, in particular to a castrated MS2 virus-like particle subunit vaccine and a preparation method thereof. Background Art

[0003] Castration is the indirect removal of an animal's reproductive system or the resulting loss of sexual function. This includes the removal of the testes in males and the ovaries in females, collectively known as gonadectomy. In addition to surgical removal of the gonads, local radiation exposure and chemical treatment can also be used for castration. Castration deprives vertebrates of their source of sex hormones, which can sometimes lead to the degeneration of genital appendages and secondary and tertiary sexual characteristics.

[0004] Gonadotropin-releasing hormone (GnRH) is an endogenous polypeptide hormone in animals. Physiological doses of GnRH-I can increase gonadotropin concentrations (e.g., a mild increase in FSH and a significant increase in LH), promote the synthesis and secretion of gonadal hormones (e.g., estradiol, progesterone, and testosterone), and promote follicular maturation and ovulation, testicular development and sperm maturation, and the development and maintenance of secondary sexual characteristics. Furthermore, GnRH-I can directly affect the gonads, regulating the synthesis and secretion of gonadal steroid hormones and promoting gamete formation.

[0005] Self-antigen proteins are often difficult to elicit antibody responses against. One approach to improving vaccination efficacy is to increase the reproducibility of the applied antigens. Unlike isolated proteins, viruses can induce rapid and potent immune responses, both with and without T cell help, without any adjuvants. Compared to a few proteins, they can trigger immune responses far more robust than their isolated components. With respect to B cell responses, it is well known that a key factor in viral immunogenicity is the reproducibility and order of surface epitopes. Many viruses exhibit quasi-crystalline surfaces with regularly arranged epitopes that efficiently crosslink epitope-specific immunoglobulins on B cells. This crosslinking of B cell surface immunoglobulins is a strong activation signal, directly inducing cell cycle progression and IgM antibody production. Furthermore, this triggered B cell activation can activate T helper cells, in turn inducing the conversion of IgM to IgG antibodies in B cells and the generation of long-lived B cell memory targets for any vaccination. Viral structure has even been implicated in the production of anti-antibodies in autoimmune diseases and is part of the natural response to pathogens. Therefore, antigens presented on highly organized viral surfaces can induce strong antibody responses against these antigens.

[0006] Virus-like particles are the main capsid protein of an RNA bacteriophage. They are self-assembled from multiple monomers into a new type of highly immunogenic virus-like particles (VLPs). These VLPs do not contain the phage RNA genome and cannot replicate. However, studies have found that a variety of polypeptides can be fused to the N- or C-terminus of the virus-like particle protein, and the resulting fusion protein forms virus-like particles when expressed in a host, typically and preferably in Escherichia coli. In addition, it has been found that if the polypeptide contains at least one antigen, the antigen or at least one antigenic site of the antigen is displayed on the outer surface of the assembled VLP, the assembled VLP can effectively improve the immunogenicity of the target antigen. In addition to the virus-like particles AP205 and Qβ that have been reported, there are also a variety of VLP platforms such as CuMV and tobacco mosaic virus VLP, all of which have the potential to be developed into castration vaccines.

[0007] Escherichia coli MS2 bacteriophage is a positively polarized, single-stranded RNA spherical virus with a 3659-bp genome encoding four proteins: a maturase, a coat protein, a replicase, and a lytic protein. Studies have shown that when the genes for the MS2 phage's maturase and coat proteins, along with the 5' noncoding sequence containing regulatory elements, are cloned into an expression vector in vitro, the proteins can self-assemble into mature virus-like particles (VLPs). Furthermore, insertion of several genes into specific sites within the coat protein gene can result in VLP expression displaying exogenous amino acids. However, there are no reports on the development of a GnRH castration vaccine using MS2.

[0008] Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a castrated MS2 virus-like particle subunit vaccine, which is easy to obtain through bacterial culture, has a high expression yield, is convenient for industrial production, has strong immunogenicity and a fast immune response.

[0010] The GNRH-I-MS2 fusion protein provided by the present invention comprises GnRH and the capsid protein of Escherichia coli MS2 phage.

[0011] The GnRH has an amino acid sequence as shown in SEQ ID NO: 1, or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO: 1; or a sequence with a homology of more than 90% to the amino acid sequence shown in SEQ ID NO: 1.

[0012] The capsid protein of the Escherichia coli MS2 phage has an amino acid sequence as shown in SEQ ID NO: 2, or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in SEQ ID NO: 2; or a sequence with a homology of more than 90% to the amino acid sequence as shown in SEQ ID NO: 2.

[0013] In the fusion protein, GnRH is located at the C-terminus or N-terminus, which is not limited by the present invention. In some embodiments, a linker is further included between GnRH and the capsid protein of Escherichia coli MS2 phage, and the linker is (G4S)n, wherein n is a non-zero integer. Preferably, n is 1.

[0014] In some specific embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3.

[0015] The present invention also provides a nucleic acid encoding the fusion protein as described above.

[0016] The nucleic acid can be DNA, RNA, cDNA or PNA. The DNA forms include cDNA, genomic DNA or artificially synthesized 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 in topology. The nucleic acid can be, for example, a part of a vector (e.g., an expression or cloning vector) or a fragment. The nucleic acid can be obtained directly from a natural source or can be prepared with the assistance of recombinant, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc. The gene sequence can use a wild-type sequence or can be codon-optimized, which is not limited by the present invention. In the present invention, the nucleic acid encoding the fusion protein is shown in SEQ ID NO: 4. The nucleic acid sequence has been codon-optimized and is more suitable for the expression of the fusion protein in the Escherichia coli system, thereby obtaining higher expression levels and biological activity.

[0017] Furthermore, the present invention also provides an expression unit comprising the nucleic acid as described above.

[0018] The expression unit includes an expression unit composed of a single or multiple nucleic acids of the present invention in tandem with a promoter and a terminator, and the present invention does not limit this.

[0019] Furthermore, the recombinant expression vector herein refers to a nucleic acid vector, a recombinant DNA molecule comprising a desired coding sequence and appropriate nucleic acid sequences or elements necessary for expression of the operably linked coding gene in a specific host organism. 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 based on the host organism. The expression vector described in the present invention can be circular or linear, and this is not a limitation of the present invention. For example, a prokaryotic host can be Escherichia coli, Bacillus, Streptomyces, or cyanobacteria; its backbone vector can be a pET series plasmid, a pGEX series plasmid, a pKBP series plasmid, or a pcDNA series plasmid. For example, the recombinant backbone vector is a pET series vector, and optionally, the backbone vector of the recombinant vector is pET-21(+), pET-24(+), pET-23(+), or pET-28a(+).

[0020] The present invention provides a method for constructing the recombinant vector, which comprises cloning the nucleic acid fragment encoding the fusion protein into the prokaryotic expression vector pET28a by homologous recombination. In some embodiments, the insertion site is between the XhoI site and the BamhI site.

[0021] Furthermore, the present invention also provides a host cell transformed or transfected with the aforementioned recombinant vector, or with the aforementioned nucleic acid integrated into its genome.

[0022] 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, and mushrooms; the yeast include Saccharomyces cerevisiae, Saccharomyces cerevisiae, Pichia pastoris and Candida, etc. The viruses include but are not limited to adenovirus, adeno-associated virus, lentivirus, and prion virus. 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 either integrated or free, and the present invention does not limit this. In the embodiment of the present invention, Escherichia coli is used as the host to express the fusion protein. The Escherichia coli is Escherichia coli BL21 (DE3).

[0023] Furthermore, the present invention also provides a culture product of the host cell.

[0024] The method for preparing the culture product or the fusion protein described above comprises culturing the host cell described above to obtain a culture product containing the fusion protein. Preferably, the host cell described above is cultured until the OD600 reaches 0.6-0.8 and IPTG is added to a final concentration of 1 mM to induce expression. After the induced expression, the supernatant is collected by centrifugation and purified by nickel column.

[0025] Furthermore, the present invention also provides a composition comprising the aforementioned fusion protein, GnRH antigen or any antigen capable of rendering animals infertile.

[0026] The composition of the present invention further comprises an acceptable adjuvant, including aluminum salt adjuvants, protein adjuvants, nucleic acid adjuvants, lipid adjuvants, mixed adjuvants, or aggregate structure adjuvants. In some embodiments of the present invention, the adjuvant is aluminum hydroxide adjuvant.

[0027] Furthermore, the fusion protein, the nucleic acid, the expression unit, the recombinant vector, the host cell, the culture product or the composition of the present invention are used in the preparation of subunit vaccines.

[0028] The vaccine is a castration vaccine. Specifically, it is a castration vaccine for male animals. The male animals are animals that can be castrated, including birds and mammals. For example, mice, rats, cats, dogs, horses, or animals of the same family and genus as the above animals. Preferably, they are felines, canines, or rodents, such as mice.

[0029] Furthermore, the present invention also provides a castrated MS2 virus-like particle subunit vaccine, which includes the fusion protein or the composition as described above.

[0030] Furthermore, the method for preparing the subunit vaccine includes mixing the fusion protein with an adjuvant; or mixing the composition with a buffer.

[0031] The present invention also provides a method for animal castration, comprising administering the aforementioned castration MS2 virus-like particle subunit vaccine, wherein the administration comprises injection.

[0032] The beneficial effects of the present invention are as follows:

[0033] First, MS2 is derived from a bacteriophage that infects Acinetobacter. Its capsid protein has the ability to self-assemble into nanoparticles, making it non-infectious and possessing strong antigenic immunity. This technology is unique to the applicant's VLP screening platform.

[0034] Second, the antigen coupled to the MS2 virus-like particles showed high antigenicity, inducing high levels of specific antibodies in mice, rats, cats, dogs, and horses, and a rapid humoral immune response;

[0035] Third, the GNRH-I-MS2 virus-like particles of the present invention can be expressed in large quantities by Escherichia coli, the production process is simple, and the GNRH-I-MS2 virus-like particles are of high purity;

[0036] Fourth, it is suitable for industrialization. The recombinant expression strain of Escherichia coli selected in the present invention has the characteristics of fast growth, easy cultivation, simple genetic manipulation, fast reproduction speed, low requirements for culture conditions, cheap culture medium, high-density culture, and tolerance to high liquid static pressure, which is convenient for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are only exemplary. For those skilled in the art, other implementation drawings can be derived from the provided drawings without inventive effort:

[0038] FIG1 is a schematic diagram of a recombinant plasmid after ligation of the GNRH-I-MS2 gene and the pET28a plasmid provided in an embodiment of the present invention;

[0039] FIG2 is a schematic diagram of SDS-PAGE running of GNRH-I-MS2 virus-like particle protein provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0040] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0042] The molecular biology experimental methods used in the examples of this invention, such as enzyme digestion and homologous recombination ligation, can be found in the second edition of Molecular Cloning. The basic materials for preparing the GNRH-I-MS2 virus-like particles of this invention include: MS2 protein nucleotide sequence, GNRH-I nucleotide sequence, pET28a plasmid, BL21(DE3) Escherichia coli strain, Tiangen Plasmid Miniprep Kit, LB medium, IPTG, kanamycin, nickel column, and imidazole. The nucleotide sequence was sent to BGI for synthesis of the GNRH-I-MS2-pET28a recombinant plasmid.

[0043] The method for preparing GNRH-I-MS2 virus-like particles expressed by recombinant Escherichia coli provided by the present invention comprises the following steps:

[0044] (1) The 10-peptide gene of GNRH-I was directly synthesized by concatenating the MS2 gene with the G4S linker and cloned into the pET28a prokaryotic expression vector to obtain the positive recombinant plasmid GNRH-I-MS2-pET28a;

[0045] (2) The recombinant plasmid obtained in step (1) was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant expression strain GNRH-I-MS2-pET28a-BL21;

[0046] (3) Cultivating the recombinant expression strain GNRH-I-MS2-pET28a-BL21 obtained in step (2), adding IPTG to induce expression, and obtaining the GNRH-I-MS2 recombinant protein.

[0047] The sequences involved in the present invention include:

[0048] The amino acid sequence of GNRH-I is: MEHWSYGLRPG (SEQ ID NO: 1)

[0049] The amino acid sequence of Linker is: GGGGS

[0050] The amino acid sequence of the MS2 protein is:

[0051] The amino acid sequence of the fusion protein is:

[0052] The nucleic acid sequence encoding the fusion protein is:

[0053] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0054] Example 1 Preparation Method of GNRH-I-MS2 Virus-Like Particles

[0055] 1 and 2 , the method for preparing bacteriophage GNRH-I-MS2 virus-like particles comprises the following steps:

[0056] (1) Construction of recombinant plasmid: The GNRH-I-MS2 gene (SEQ ID NO: 4) was directly synthesized by BGI into the pET28a vector at the XhoI and BamhI restriction sites to obtain the recombinant plasmid GNRH-I-MS2-pET28a;

[0057] (2) Transforming the recombinant plasmid into an expression strain: The recombinant plasmid GNRH-I-MS2-pET28a was transformed into an Escherichia coli BL21 (DE3) expression strain to obtain a GNRH-I-MS2-pET28a-BL21 recombinant expression strain;

[0058] (3) Bacterial culture and purification of GNRH-I-MS2 virus-like particles: The GNRH-I-MS2-pET28a-BL21 recombinant expression strain was cultured and IPTG-induced expression was used to obtain GNRH-I-MS2 virus-like particles.

[0059] Specifically, the steps for expressing and purifying GNRH-I-MS2 virus-like particles are as follows:

[0060] (a1) Preparation of GNRH-I-MS2-pET28a recombinant plasmid

[0061] Use a pipette to inoculate 5 μl of the glycerol stock containing the plasmid into 5 mL of 2YT medium (containing 50 μg / mL kanamycin). Incubate at 37°C with shaking for 14-16 hours. After incubation, remove 1 mL of the culture medium for sequencing. Use a plasmid extraction kit to extract the plasmid from the remaining culture medium, and measure the nucleic acid concentration using a protein nucleic acid detector.

[0062] (a2) GNRH-I-MS2 virus-like particle expression

[0063] Transform the BL21(DE3) expression strain, shake the cells, concentrate by centrifugation, spread on kanamycin-resistant plates, and incubate overnight at 37°C. Scrape a colony and inoculate 10 mL of LB medium containing kanamycin. Incubate at 37°C at 180 rpm until the OD value is approximately 0.8. Transfer the colony to 500 mL of LB medium containing kanamycin and shake at 37°C at 180 rpm until the OD value is approximately 0.8. Add inducer (1 mM IPTG) to a final concentration of 1 mM, and express overnight at 30°C. On the third day, harvest the cells by centrifugation at 4500 rpm for 15 minutes. Disrupt the cells by sonication for 45 minutes, 3 seconds on, 4 seconds off, at 125 W. After sonication, collect the supernatant at 12000 rpm for 10 minutes at 4°C.

[0064] (a3) Purification of GNRH-I-MS2 virus-like particles

[0065] The supernatant was centrifuged and purified by passing it through a nickel column. The column was equilibrated with PBS for 5 column volumes. Sample was loaded, washed with PBS for 5 column volumes, then washed with 25 mM imidazole for 2 column volumes, and then washed again with 25 mM imidazole for 5 column volumes. Elution was then performed with 50, 100, 150, 200, and 250 mM imidazole for 5 column volumes. The flow-through from sample loading to 500 mM imidazole was collected.

[0066] Aspirate 10 μL of each collected solution, add 10 μL of 2× protein electrophoresis loading buffer, and heat at 100°C for 4 minutes. Then, perform protein electrophoresis and stain to determine the concentration of the target band in the collected solution. Ultrafilter the target protein using a 3 kDa ultrafiltration tube and replace the solvent with PBS.

[0067] Example 2 GNRH-I-MS2 virus-like particle subunit vaccine immunization method

[0068] As described in Example 1, GNRH-I-MS2 virus-like particles were mixed with aluminum hydroxide adjuvant for immunization. Eight-week-old male C57BV / 6 mice (five mice per group) were immunized with 50 μg of GNRH-I-MS2 virus-like particle vaccine on days 0, 14, and 28. Groups of GNRH-I-MS2 without adjuvant, adjuvant-PBS-negative, MS2-only, and GNRH-I-only were also set up. Anti-GNRH-I recombinant protein antibody titers and testosterone levels were measured in these mice. On day 70 after immunization, the mice were sacrificed and testicular weights were measured.

[0069] Example 3 Determination of mouse anti-GNRH-I antibody titer

[0070] At various time points during the experiment, serum was collected from immunized and control mice. Anti-GNRH-I IgG antibody titers were determined by ELISA as follows. 96-well plates were coated with 2 μg / ml GnRH-I-BSA overnight at 4°C, with 100 μl added to each well. The next day, the plates were washed five times with 1 / 1000 PBST and blocked with 300 μl of 2% BSA at 37°C for 2 h. The plates were then washed five times with 1 / 1000 PBST. Mouse serum was serially diluted from the initial dilution (1:500), followed by 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000 in 2% BSA. From the highest to the lowest dilution, 100 μl was added to each well and incubated at room temperature with shaking for 45 min. Wash the plate five times with 1 / 1000 PBST. Use HRP-conjugated goat anti-mouse polyclonal antibody as a secondary antibody at a dilution of 1:5000 in 3% skim milk, with 100 μl per well, and incubate at room temperature with shaking for 45 minutes. Develop with TMB for 5-10 minutes, stop with 2M sulfuric acid, and read directly at 450 nm. Determine the optical density (OD) at 450 nm using an ELISA reader (BioRad Benchmark). Use these data to calculate the maximum OD450 serum dilution.

[0071] Table 1 shows that in male mice immunized with GNRH-I-MS2 virus-like particles without adjuvant, the average titer reached 8,000 on day 28, and after the third booster dose, the average titer remained at 64,000 and then maintained at 32,000. The average titer of the GNRH-I-MS2 virus-like particle adjuvant group reached 64,000 on day 28 and then remained at 256,000 after the third dose. In contrast, the titer of the GNRH-I alone immunization group was only 16,000 on day 28. These results clearly indicate that GNRH-I-MS2 virus-like particles can induce high anti-GNRH antibody titers, and the immunization effect is even better with the addition of an adjuvant.

[0072] Table 1

[0073] At various time points during the above experiments, serum was collected from immunized mice and control mice. Testosterone levels in mouse serum were determined using Testosterone-ELISA (IBL, Hamburg, Germany).

[0074] Table 2 shows that in mice immunized with GNRH-I-MS2 virus-like particles without aluminum hydroxide adjuvant, mean testosterone levels were significantly suppressed (<2 ng / ml) on day 42 after immunization, and levels remained below 2 ng / ml on day 70. In mice immunized with GNRH-I-MS2 supplemented with aluminum hydroxide, mean testosterone levels dropped to <2 ng / ml on day 28. The mean central nervous system hormone level was approximately 38-fold lower than that in the control group of mice immunized with GNRH-I plus aluminum hydroxide. This clearly demonstrates the neutralizing activity of the induced antibody response.

[0075] Table 2 Testosterone levels in mouse serum

[0076] Mice were sacrificed on day 70, and the testicles were removed and weighed before being fixed in 4% formaldehyde.

[0077] Table 3 shows that the testicular weight of mice immunized with GnRH-I did not decrease on day 70, while the testicular weight of mice receiving GNRH-I-MS2 virus-like particles and aluminum hydroxide adjuvant was reduced by 76.96% compared with the control group, which clearly indicated the neutralizing activity of the induced antibody response.

[0078] Table 3 Testis weight

[0079] Safety study of GNRH-I-MS2 virus-like particle subunit vaccine in mice

[0080] The GNRH-I-MS2 virus-like particle vaccine was subcutaneously injected into 5 mice at a dose of 0.5 ml each. After 7 days of observation, the mice showed no obvious local or systemic reactions or deaths caused by the vaccine, indicating that the prepared GNRH-I-MS2 virus-like particle subunit vaccine is safe.

Claims

1. GNRH-I-MS2 fusion protein, characterized in that Includes GnRH and the capsid protein of E. coli MS2 phage; wherein GnRH has an amino acid sequence as shown in SEQ ID NO: 1; The capsid protein of Escherichia coli MS2 phage has the amino acid sequence shown in SEQ ID NO:

2.

2. The fusion protein according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

3.

3. A nucleic acid encoding the fusion protein according to claim 1 or 2.

4. An expression unit comprising the nucleic acid according to claim 3.

5. A recombinant vector comprising the nucleic acid according to claim 3 or the expression unit according to claim 4.

6. A host cell transformed or transfected with the recombinant vector of claim 5, or a host cell in which the nucleic acid of claim 3 is integrated into its genome.

7. A culture product of the host cell according to claim 6.

8. The method for preparing the fusion protein according to claim 1 or 2, characterized in that: The method comprises culturing the host cell described in claim 6 to obtain a culture product containing the fusion protein described in claim 1 or 2.

9. A vaccine, characterized in that Comprising the culture product according to claim 7 and an adjuvant.

10. The method for preparing the vaccine according to claim 9, characterized in that: The culture product according to claim 7 is purified and mixed with an adjuvant.

11. A method for castration of an animal, characterized in that Comprising administering the vaccine of claim 10.

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