Recombinant protein, vaccine and application of Clostridium perfringens type G

Abstract

This application provides a recombinant protein, vaccine, and application of Clostridium perfringens type G. Specifically, the recombinant protein of Clostridium perfringens type G has the amino acid sequence set forth in SEQ ID NO. 3. Immunization with this recombinant protein can effectively prevent and treat necrotic enteritis in chickens caused by Clostridium perfringens type G. The vaccine is safe, reliable, and simple to prepare.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a recombinant protein, vaccine and application of type G Clostridium perfringens. Background Art

[0002] Clostridium perfringens is widely distributed in the intestines of humans, animals, and birds. It can infect many tissues in humans and animals, causing enterotoxic disease. The bacterium's gliding motility is associated with its toxic function and contributes to its adhesion and biofilm formation.

[0003] The toxicity of Clostridium perfringens can be attributed to its possession of over 20 potent toxins and hydrolases. Different strains of C. perfringens can produce a variety of exotoxins and invasive enzymes, depending on their toxin production patterns. Based on the type of toxins produced, strains can be divided into seven toxinotypes (A, B, C, D, E, F, and G). Type G is the primary pathogen of necrotic enteritis (NE) in poultry.

[0004] Currently, the poultry industry primarily treats necrotic enteritis with antibiotics. However, with increasing reports of bacteria becoming resistant to antibiotics, a safe and effective method for preventing necrotic enteritis in poultry is urgently needed. Summary of the Invention

[0005] Based on this, the present application provides a recombinant protein, vaccine and application of Clostridium perfringens type G that can effectively prevent and treat necrotic enteritis in poultry.

[0006] According to one aspect of the present application, a recombinant protein of Clostridium perfringens type G is provided, wherein the recombinant protein has an amino acid sequence as shown in SEQ ID NO.3.

[0007] An isolated nucleic acid encoding the above-mentioned recombinant protein.

[0008] In one embodiment, the sequence of the nucleic acid is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0009] A recombinant vector contains the above nucleic acid.

[0010] A host cell, the genome of which is incorporated with the above-mentioned nucleic acid or the above-mentioned recombinant vector.

[0011] In one embodiment, the host cell is a prokaryotic cell.

[0012] A vaccine comprising the above-mentioned recombinant protein, the above-mentioned nucleic acid or the above-mentioned recombinant vector.

[0013] In one embodiment, an adjuvant is also included.

[0014] A complete kit comprises the above-mentioned vaccine and a container for vaccinating the vaccine.

[0015] According to another aspect of the present application, provided is the use of the above-mentioned recombinant protein, the above-mentioned nucleic acid or the above-mentioned recombinant vector in the preparation of a drug for preventing and treating G-type Clostridium perfringens infection.

[0016] Compared with traditional technologies, this application has the following beneficial effects:

[0017] The present application develops a recombinant protein with the amino acid sequence shown in SEQ ID NO. 3 targeting the α-toxin and NetB toxin of Clostridium perfringens type G. This recombinant protein can produce effective immune protection against Clostridium perfringens type G and is safe, stable, and has no toxic side effects.

[0018] In addition, the recombinant protein of the present application can be used in the form of molecular vaccine or oral vaccine to effectively prevent and treat poultry necrotic enteritis caused by Clostridium perfringens type G. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0020] Figure 1 This is the recombinant plasmid map constructed in Example 2 of this application;

[0021] Figure 2 This is the gel electrophoresis diagram of the colony PCR identification in Example 2 of the present application;

[0022] Figure 3 This is a diagram showing the purification and identification results of the recombinant protein in Example 2 of the present application;

[0023] Figure 4 This is the recombinant plasmid map constructed in Example 3 of this application;

[0024] Figure 5 This is the gel electrophoresis diagram of the colony PCR identification in Example 3 of the present application;

[0025] Figure 6 This is a diagram showing the results of intestinal lesions in chickens in Example 4 of this application. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotations of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the technical field of this application. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or can be prepared by existing methods.

[0028] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0029] Some embodiments of the present application provide a recombinant protein of Clostridium perfringens type G, which has an amino acid sequence as shown in SEQ ID NO.3.

[0030] Specifically, the amino acid sequence shown in SEQ ID NO.3 is:

[0031] MGSDPSVGNNVKELVAYISTSGEKDAGTDDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWEAAAKYYGK MKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGLE

[0032] The present application also provides an isolated nucleic acid encoding the above-mentioned recombinant protein.

[0033] The nucleic acid can be RNA or DNA.

[0034] In some embodiments, the nucleotide sequence of the nucleic acid is codon-optimized for different host cells.

[0035] Codon optimization refers to optimizing the target gene by using the preferred codons of the host cell without changing the amino acid sequence, thereby improving the expression efficiency of the recombinant protein.

[0036] In some embodiments, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0037] In addition, the amino acid sequence of the recombinant protein can also be a sequence substantially similar to the amino acid sequence selected from SEQ ID NO: 3. The nucleotide sequence of the nucleic acid can also be a sequence substantially similar to the nucleotide sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2. "Substantially similar" means that the given nucleic acid or amino acid sequence shares at least 95% identity with the reference sequence, for example, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. Alternatively, it means that the given nucleic acid or amino acid sequence differs from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid or amino acid residues. For polypeptides, such differences are preferably amino acid substitutions or deletions. Preferably, the substantially similar sequence also retains the unique activity of the polypeptide that can efficiently detect endogenous antibodies. Generally, substitutions are considered conservative substitutions, such as substitutions between the aliphatic amino acids Ala, Val, Leu, and Ile, interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitutions between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg, and substitutions between the aromatic residues Phe and Tyr.

[0038] A "substantially similar" amino acid sequence can also be a derivative of the polypeptide shown in SEQ ID NO.3. The term "derivative" refers to a chemically modified protein or polypeptide that has been chemically modified by natural processes (such as processing and other post-translational modifications) or by chemical modification techniques, for example, by the addition of one or more polyethylene glycol molecules, sugars, phosphates and / or other such molecules, one or more of which are not naturally attached to the wild-type protein. Derivatives include salts. Such chemical modifications are described in detail in basic textbooks and more detailed monographs, as well as in a large amount of research literature, and are well known to those skilled in the art. It should be understood that the same type of modification can be present at several sites in a given protein or polypeptide to the same or varying degrees. In addition, a given protein or polypeptide can contain many types of modifications. Modifications can occur at any position in the protein or polypeptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cysteine ​​formation, pyroglutamate formation, methylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, protein enzymatic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation and ADP-ribosylation, selenization, sulfation, transfer RNA-mediated addition of amino acids of proteins (such as arginylation) and ubiquitination. They can also be conjugated to vitamins, such as biotin, folic acid or vitamin B12.

[0039] The present application also relates to a recombinant vector containing the above-mentioned nucleic acid.

[0040] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). In some embodiments, the vector described in the present application contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0041] In some embodiments, the vectors described herein may further comprise a gene for screening (e.g., an antibiotic resistance gene), a nucleic acid fragment for producing a fluorescent protein, and the like. The fluorescent protein may be green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein.

[0042] Green fluorescent protein can be the common GFP, or a modified GFP gene, such as the enhanced GFP gene EGFP; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP, etc.; orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate, etc.

[0043] The present application also relates to a host cell, the genome of which is incorporated with the above-mentioned nucleic acid or the above-mentioned vector.

[0044] In some embodiments, the host cells of the present application are transformed with the above-mentioned vectors.

[0045] The term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, Lactobacillus, or Bacillus subtilis; fungal cells such as yeast cells or Aspergillus; insect cells such as Drosophila S2 cells or Sf9 cells; or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK-293 cells, or human cells. Host cells are generally not totipotent animal cells (preferably avian cells, such as chicken cells), and for example, do not include fertilized eggs, embryos, germline stem cells, or embryonic stem cells. Preferably, the host cell is a prokaryotic cell; more preferably, Escherichia coli or Lactobacillus.

[0046] The present application also relates to a vaccine comprising the above-mentioned recombinant polypeptide, the above-mentioned nucleic acid or the above-mentioned vector.

[0047] In some embodiments, the above-mentioned vaccine further comprises an adjuvant. Adjuvants suitable for use in the vaccines of the present application include adjuvants that can enhance or amplify antibody immune responses, as well as adjuvants that can enhance cell-mediated T cell epitope responses. These adjuvants are well known in the art.

[0048] In some embodiments, the adjuvant is selected from one or more of alum, complete Freund's adjuvant, incomplete Freund's adjuvant, squalene, squalane, muramyl dipeptide, MF59, AS03, monophosphatidyl lipid A, flagellin, CpG-ODN, poly(I:C), and small molecules such as aluminum or calcium salts. These adjuvants are well known in the art and available through several commercial channels. Of these, complete Freund's adjuvant, incomplete Freund's adjuvant, squalane, and alum are generally not used in humans. When added, the amount of adjuvant in the vaccine is typically between about 1% (v / v) and 20% (v / v). In specific embodiments, the amount of adjuvant is between about 2% (v / v) and 10% (v / v). In more specific embodiments, the amount of adjuvant is between about 3% (v / v) and 6% (v / v).

[0049] In some embodiments, the above-mentioned vaccine further includes a pharmaceutically acceptable carrier.

[0050] "Pharmaceutically acceptable carrier" is intended to aid in the stabilization and administration of the vaccine while being harmless and well tolerated by the target. Such carriers may, for example, be sterile water or sterile physiological saline solutions. In more complex forms, the carrier may, for example, be a buffer solution, which may contain further additives, such as stabilizers or preservatives. Water or aqueous saline solutions and aqueous sugar (e.g., dextrose and / or glycerol) solutions may be used as carriers, particularly for injectable solutions. In addition, the carrier may be and / or comprise a hydrocolloid and / or polymer solution, for example, to thicken the poultry vaccine to be sprayed on the poultry.

[0051] In some embodiments, the vaccine is a water-in-oil emulsion having an aqueous phase and an oily phase.

[0052] In some embodiments, the vaccine is an oil-in-water emulsion having an aqueous phase and an oily phase.

[0053] Furthermore, the above vaccines comprise inactivated viruses and / or inactivated bacteria (e.g. bacterins) and / or antigens of bacterins. This can be derived from the microorganism pathogenic to poultry in any suitable manner, e.g. as "live" attenuated, inactivated or subunit antigens.

[0054] Typical immunizations are by subcutaneous (SC) or intramuscular (IM) injection, or the vaccines may be administered via a skin patch, in a delayed-release implant, scarification, or topical application. Administration may also be via the recipient bird's drinking water and / or food.

[0055] The present application also relates to a complete kit comprising the above-mentioned vaccine and a container for administering the vaccine.

[0056] The present application also relates to a method for preparing the above-mentioned vaccine, comprising the following steps:

[0057] The host cells are cultured under appropriate conditions, the culture fluid and / or host cell lysate are collected, and then separated and purified to prepare the vaccine.

[0058] The present application also relates to the use of the above-mentioned recombinant protein, the above-mentioned nucleic acid or the above-mentioned recombinant vector in the preparation of a drug for preventing and treating G-type Clostridium perfringens infection.

[0059] The present application further provides a method for protecting poultry from infection by Clostridium perfringens type G, which comprises administering a preventively effective amount / therapeutically effective amount of the vaccine of the present application to the poultry.

[0060] The term "poultry" refers to wild or domesticated chickens, ducks, geese, swans, geese, pigeons, quails and other birds, especially chickens.

[0061] Factors that influence the preferred dosage regimen may include, for example, the species or breed (e.g., avian species or breed), age, weight, diet, activity, lung size, and condition of the subject; the route of administration; the efficacy, safety, and duration of immunity profile of the specific vaccine used; whether a delivery system is used; and whether the vaccine is administered as part of a drug and / or vaccine combination. Thus, the dosage actually employed may vary for a particular animal and, therefore, may deviate from the typical dosages described above. Determination of such dosage adjustments is generally within the skill of one skilled in the art of vaccine development using conventional methods.

[0062] The present application will be further described below with reference to specific embodiments and comparative examples, but they should not be construed as limiting the scope of protection of the present application.

[0063] Example 1: Screening of coding genes

[0064] Targeting the α-toxin and NetB toxin of type G Clostridium perfringens, an amino acid sequence as shown in SEQ ID NO.3 was obtained by screening.

[0065] The amino acid sequence shown in SEQ ID NO.3 is:

[0066] MGSDPSVGNNVKELVAYISTSGEKDAGTDDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWEAAAKYYGK MKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGLE

[0067] Codon optimization was performed for different host cells to obtain the coding gene sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0068] The nucleotide sequence shown in SEQ ID NO.1 is:

[0069] CATATGGGCAGCGATCCGAGCGTGGGTAATAATGTGAAAGAACTGGTTGCCTATATTAGCACCAGCGGTGAAAAAGATGCAGGTACCGATGATTATATGTATTTTGGTATTAAGACCAAGGACGGTAAAACCCAGGAATGGGAAATGGATAATCCGGGTAATGATTTTATGGCCGGTAGCAAAGATACCTATACCTTTAAACTGAAAGACGAAAATCTGAAAATCGATGATATCCAGAATATGTGGATTCGCAAACGCAAATATACCGCCTTTCCGGATGCCTATAAACCGGAAAATATTAAAGTGATTGCGAATGGTAAGGTTGTGGTTGATAAAGATATTAATGAGTGGGAAGCCGCCGCAAAATATTATGGTAAAATGAAATGGCCGGAAACCTATCGTATTAATGTTAAAAGTGCAGATGTGAATAACAACATTAAGATTGCAAACAGTATCCCGAAAAATACCATTGATAAAAAGGATGTTAGCAACAGCATTGGTTATAGTATTGGCGGTAATATTAGCGTGGAAGGTAAAACCGCAGGTGCCGGTATTAATGCAAGCTATAATGTTCAGAATACCATTAGTTACGAGCAGCCGGATTTTCGTACCATTCAGCGCAAAGATGATGCAAATCTGGCCAGTTGGGATATTAAATTTGTGGAAACCAAAGATGGTTACAATATTGATAGCTATCACGCCATTTATGGCAATCAGCTGTTTATGAAAAGCCGTCTGTATAATAATGGTCTCGAG

[0070] The nucleotide sequence shown in SEQ ID NO.2 is as follows:

[0071] CCATGGGAATTCAGATCTTATGCTTTTGTTATAAGTTAGCACAAAAAAGCAGAAAATAAAAAGTAGAAATAAAAAAAGATGTTTTTTTGCCCATATCTCTATGAAAAAAACTGTGAAATGTGTAAAATATGGATGAAACATTGAATTTAAAAGGAGATATTTCATGAAGAAGGAATTGTCATTCCATGAAAAATTGTTGAAG

[0072] TTGACTAAGCAACAAAAGAAAAAGACTAACAAGCATGTTTTCATTGCTATT

[0073] CCAATTGTTTTCGTTTTGATGTTTGCTTTTATGTGGGCTGGTAAAGCTGAAA

[0074] CTCCCAAGTTAAGACTTATTCAGATGATGTTTTGTCAGCTTCATTTGTTGG

[0075] TGATATTATGATGGGTCGTTATGTTGAAAAGGTTACTGAACAAGGGTGC

[0076] TGATTCAATTTTTCAATACGTTGAACCAATTTTCCGTGCTTCAGATTATGTTG

[0077] CTGGTAATTTTGAAAACCCAGTTACTTATCAAGGAACTATAAGCAAGCTG

[0078] ATAAGGAAATTCATTTGCAAACTAATAAGGAAAGCGTTAAGGTTTTAAAGG

[0079] ATATGAATTTCACTGTTTTGAACTCAGCTAATAACCATGCTATGGATTATGGT

[0080] GTTCAAGGTATGAAGGATACTTTAGGTGAATTTGCTAAGCAAAATTTGGATA

[0081] TTGTTGGTGCTGGTTATTCATTATCAGATGCTAAAAAAAGATTAGCTACCA

[0082] AAAGGTTAACGGTGTTACTATTGCTACTTTAGGTTTTACTGATGTTTCAGGT

[0083] AAGGGTTTTGCTGCTAAGAAGAATACTCCAGGTGTTTTACCAGCTGATCCA

[0084] GAAATTTTTATTCCAATGATTAGCGAAGCTAAGAAACATGCTGATATTGTTG

[0085] TTGTTCAATCACATTGGGGTCAAGAATATGATAATGATCCAAATGATCGTCA

[0086] ACGTCAATTAGCTCGTGCTATGTCAGATGCTGGTGCTGATATTATTGTTGGT

[0087] CATCATCCACATGTTTTAGAACCAATTGAAGTTTATAACGGTACTGTTATTTT

[0088] CTACTCATTGGGTAATTTCGTTTTCGATCAAGGTTGGACTCGTACTCGTGAT

[0089] TCAGCTTTAGTTCAATATCATTTAAAGAAGAACGGTACTGGTCGTTTTGAA

[0090] GTTACTCCAATTGATATTCATGAAGCTACTCCAGCTCCAGTTAAGAAGGATT

[0091] CATTAAAGCAAAAGACTATTATTCGTGAATTGACTAAGGATTCAAACTTTG

[0092] CTTGGAAAGTTGAAGATGGTAAGTTAACTTTTGACATTGATCATTCAGACA

[0093] AGTTAAAGTCAAAGGAAGGTAAGTCATCAGGTTCAGGTTCAGAATCAAAA

[0094] TCAACTGGATCCGCTCCACCACATGCTTTATCAGGTTCAGATCCATCAGTTG

[0095] GTAATAATGTTAAGGAATTAGTTGCTTACATTAGCACTTCAGGTGAAAAAAG

[0096] ATGCTGGTACTGATGATTATATGTATTTTGGTATTAAAGACTAAGGACGGTAA

[0097] AACTCAAGAATGGGAAAATGGATAATCCAGGTAATGATTTTATGGCTGGTTC

[0098] AAAAGATACTTTATACTTTTAAGTTGAAGGACGAAAACTTGAAAATTGATGA

[0099] TATTCAAAACATGTGGATTCGTAAACGTAAATATACTGCTTTTCCAGATGCT

[0100] TATAAGCCAGAAAATATTAAAGTTATTGCTAACGGTAAGGTTGTTGTTGATA

[0101] AAGATATTAACGAATGGGAAGCTGCTGCTAAATATTATGGTAAAATGAAGT

[0102] GGCCAGAAACTTATCGTATTAATGTTAAGTCAGCTGATGTTAATAACAACAT

[0103] TAAGATTGCTAACAGCATTCCAAAAAAACACTATTGATAAGAAGGACGTTTC

[0104] AAATTCAATTGGTTATTCAATTGGTGGTAACATTTCAGTTGAAGGTAAAACT

[0105] GCTGGTGCTGGTTATTAATGCTTCATATAATGTTCAAAAACACTATTAGCTACG

[0106] AACAACCAGATTTTCGTACTATTCAACGTAAAGATGATGCTAATTTGGCTTC

[0107] ATGGGATATTAAGTTTGTTGAAACTAAGGATGGTTACAATATTGATTCATAC

[0108] CATGCTATTTACGGTAATCAATTATTCATGAAGAGCCGTTTATACAATAACG

[0109] GTGGATCCGAGCTCGGGCCCTCTAGAGTCGACCTCGAGCACCACCACCAC

[0110] CACCACTGATAACCCGGGTAATCTGAAGAAAAAGGAGGCTAGTATACTAGC

[0111] CTCCTTTTCTTCAGATTACCCGGGGCGGCCGCAAGCTT

[0112] Example 2: Preparation of recombinant protein

[0113] (1) The nucleotide sequence obtained by artificial screening and combination, such as the gene fragment shown in SEQ ID NO.1, was synthesized, and then double-digested with NdeI and XhoI restriction endonucleases with the prokaryotic expression vector pET-30a(+), and the nucleic acid fragments were recovered and ligated overnight with T4 DNA ligase; the recombinant plasmid map after ligation was as shown in FIG. Figure 1 shown.

[0114] (2) The ligation product was heat-shock transformed into BL21 (DE3) infected cells and antibiotic pressure screening was performed using LB culture plates containing 50 μg / ml kanamycin. The cells were identified by colony PCR (e.g. Figure 2 After nucleic acid sequencing analysis, a recombinant bacterium was obtained. The bacterium contained the recombinant plasmid constructed in step (1).

[0115] (3) Pick a single colony of the recombinant bacteria screened in step (2) and place it in 5 mL of LB liquid medium containing 50 μg / ml kanamycin. After overnight culture, inoculate it into 100 mL of LB liquid medium containing 50 μg / ml kanamycin at a 1% inoculum size. Cultivate it at 25°C until the logarithmic phase. Add 1 mM IPTG inducer and continue culturing for 16 hours before collecting the bacteria. Add PBS buffer to the above bacteria, wash it twice in a row, add 10 mL of PBS buffer containing 1% Triton X-100, ultrasonically lyse the bacteria on ice, centrifuge at 12000 rpm for 20 minutes, and collect the supernatant Q1. Use Ni-Sepharose affinity chromatography column to separate and purify the target protein in the supernatant Q1, collect the eluate, and obtain the recombinant protein of Clostridium perfringens type G.

[0116] The above results show that the recombinant bacteria can effectively express the target protein after induction, with a size of about 35KDa. The purification results of the recombinant protein are as follows Figure 3 shown.

[0117] Example 3: Preparation of oral vaccine

[0118] (1) A codon-optimized gene fragment (nucleotide sequence shown in SEQ ID NO.2) was artificially synthesized, and then fragment 1 was cloned using primers with Not I restriction site (nucleotide sequence shown in SEQ ID NO.4) and Hind III restriction site (nucleotide sequence shown in SEQ ID NO.5). At the same time, fragment 2 was cloned using primers with BamH I restriction site (nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7). The above two fragments were sequentially digested and ligated into the pNZ8148 vector, and correct positive clones were screened by colony PCR to obtain plasmid 8148-CACN. The plasmid map is shown in FIG. Figure 4 shown.

[0119] Table 1 Primer information

[0120]

[0121]

[0122] (2) Prepare NZ9000 competent cells. First, streak the NZ9000 bacterial liquid on GM17 solid medium and culture it at 30℃ for 24h~36h under anaerobic conditions; pick the single colony after streaking the plate into 5mL GM17 liquid medium and culture it at 30℃ for 12h~16h under anaerobic conditions; inoculate it into 50mL fresh G / L-SGM17 liquid medium at a ratio of 5% and culture it at 30℃ under anaerobic conditions until OD 600Reach 0.3-0.4 (estimated 2h-3h); centrifuge at 4°C, 5000g for 10min, discard the supernatant, and retain the bacterial pellet; add 5mL of washing solution III to the bacterial pellet, resuspend the bacteria, and let it stand at room temperature for 30min; centrifuge at 4°C, 5000g for 10min, discard the supernatant, and retain the bacterial pellet; add 5mL of washing solution I (pre-cooled) to the bacterial pellet, resuspend the bacteria; centrifuge at 4°C, 5000g for 10min, discard the supernatant, and retain the bacterial pellet; add 5mL of washing solution II (pre-cooled) to the bacterial pellet, resuspend the bacteria; centrifuge at 4°C, 5000g for 10min, discard the supernatant, and retain the bacterial pellet; add 1mL (1 / 50 culture volume) of washing solution I (pre-cooled) to the bacterial pellet, resuspend the bacteria, and aliquot the competent cells (ice bath) at 100μL / tube, and finally store in a -80°C refrigerator for use.

[0123] Among them, washing solution I includes 0.5 mol / L sucrose and 10% glycerol; washing solution II includes 0.5 mol / L sucrose, 10% glycerol and 50 mmol / L EDTA; washing solution III includes 100 mmol / L lithium acetate dihydrate, 10 mmol / L dithiothreitol (DTT), 0.6 mol / L sucrose and 1 mol / L Tris-HCl (pH 7.5).

[0124] (3) Add 400 ng of the plasmid 8148-CACN prepared in step (1) to 100 μL of NZ9000 competent cells, mix well and place on ice for 5 minutes; quickly transfer to a 2 mm electric shock cup (pre-cooled for more than 10 minutes), place in an electroporator, and discharge at 2.5 kV with a discharge duration of 4.5 ms to 5 ms; quickly add 900 μL of recovery medium (pre-cooled) after electroporation, and ice bath for 5 minutes; transfer the electroporation product to a 1.5 mL centrifuge tube and incubate at 30°C for 2 hours; take 100 μL and apply it to GM17 solid culture medium containing 1 / 2000 and 1 / 4000 chloramphenicol antibiotics, and incubate at 30°C for 36 hours to 48 hours under anaerobic conditions; pick the transformants for colony PCR identification, and the results are as follows Figure 5 As shown, colonies of the expected size were obtained; an oral vaccine was thus prepared.

[0125] In this example, the nucleotide sequence encoding the recombinant protein of Clostridium perfringens type G was connected to an expression vector, and the protein was expressed on the surface of Lactobacillus cells through pgsA (polycobalamin synthase A), thereby preparing an oral vaccine.

[0126] Example 4: Animal Experimental Evaluation of Clostridium perfringens Type G Vaccine

[0127] (1) One-day-old unvaccinated 817 broiler chickens were selected and divided into three groups, with 30 test chickens in each group. All test chickens were kept in cages without coccidia and had free access to water and food. Among them, the immunized group was subcutaneously injected with the recombinant protein prepared in Example 2 at 1 day, 7 days, and 14 days of age. Thereafter, both the immunized group and the positive control group were challenged with Clostridium perfringens type G from 26 to 29 days of age, at a rate of 8×10 8 CFU / perform only.

[0128] The rats were weighed at 1 day, 21 days, and 30 days of age, and the average weight gain and relative weight gain rate were calculated. At 30 days of age, the survival rate was calculated. The results are shown in Table 2.

[0129] Survival rate = number of surviving chickens at 30 days of age / number of surviving chickens at 21 days of age * 100%

[0130] Average weight gain (g) = average weight at 30 days of age (g) - average weight at 21 days of age (g)

[0131] Relative weight gain rate (%) = (average weight gain of infected chickens / average weight gain of uninfected chickens) × 100%

[0132] Table 2

[0133] Experimental group Relative weight gain rate Survival rate Immunization group 102％ 97％ Positive group 81％ 97％ Negative group 100％ 100％

[0134] (2) Some chickens were killed and the duodenum, jejunum, ileum and cecum were collected for lesion scoring and photographic recording. The results are shown in Table 3 and Figure 6 shown.

[0135] Scoring criteria are as follows: 0 = no obvious lesions, 1 = intestinal mucosal congestion, 2 = small focal necrosis or ulceration (1-5 lesions), 3 = focal necrosis or ulceration (6-15 lesions), 4 = focal necrosis or ulceration (16 or more lesions)

[0136] Table 3

[0137] Experimental group duodenum jejunum cecum Immunization group 1.35 0.76 0.18 Positive group 2.42 1.00 0.68 Negative group 1.45 0.50 0.15

[0138] It can be seen that after immunizing chickens with the recombinant protein of the present application, the intestinal lesions were significantly improved compared with the positive control group, while the relative weight gain rate and survival rate were similar to those of the negative control group, indicating that the recombinant protein of the present application can effectively improve the intestinal damage caused by type G Clostridium perfringens.

[0139] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0140] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A recombinant protein of Clostridium perfringens type G, characterized in that The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

3.

2. An isolated nucleic acid, characterized in that Encoding the recombinant protein according to claim 1.

3. A recombinant vector, characterized in that Containing the nucleic acid according to claim 2.

4. A host cell, characterized in that The nucleic acid according to claim 2 or the recombinant vector according to claim 3 is incorporated into its genome.

5. The host cell according to claim 4, characterized in that The host cell is a prokaryotic cell.

6. A vaccine, characterized in that Contains the recombinant protein according to claim 1.

7. The vaccine according to claim 6, characterized in that Adjuvants are also included.

8. A complete kit, characterized in that: Comprising the vaccine according to any one of claims 6 to 7, and a container for inoculating the vaccine.

9. Use of the recombinant protein according to claim 1, the nucleic acid according to claim 2, or the recombinant vector according to claim 3 in the preparation of a medicament for preventing and treating G-type Clostridium perfringens infection.

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