CN116284281A

CN116284281A - Recombinant protein of clostridium perfringens G, vaccine and application

Info

Publication number: CN116284281A
Application number: CN202310209976.9A
Authority: CN
Inventors: 蔡海明; 胡俊菁; 申翰钦; 孙铭飞; 廖申权; 戚南山; 李娟�; 吕敏娜; 林栩慧; 宋勇乐; 肖文婉; 张小慧; 张健騑
Original assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority date: 2023-03-07
Filing date: 2023-03-07
Publication date: 2023-06-23

Abstract

The application provides a recombinant protein of clostridium perfringens G, a vaccine and application thereof. Specifically, the recombinant protein of clostridium perfringens type G has an amino acid sequence shown in SEQ ID No. 3. Through immunization of the recombinant protein, necrotic enteritis of chickens caused by clostridium perfringens G can be effectively prevented; and has the characteristics of safety, reliability, simple preparation method and the like.

Description

Recombinant protein of clostridium perfringens G, vaccine and application

Technical Field

The application relates to the field of biotechnology, in particular to a recombinant protein of clostridium perfringens type G, a vaccine and application.

Background

Clostridium perfringens (Clostridium perfringens) is widely distributed in the intestinal tract of humans, animals and birds. Clostridium perfringens can infect many tissues of humans and animals, causing enterotoxigenic diseases; the sliding movement of the bacteria is related to its toxic function, contributing to the adhesion capacity of the strain and to the biofilm formation.

The toxicity of clostridium perfringens can be attributed to its possession of more than 20 strong toxins and hydrolases. Clostridium perfringens can produce a variety of exotoxins and invasive enzymes depending on the mode of toxin production of the different strains, and the strains can be classified into seven toxin types (type a, type B, type C, type D, type E, type F, and type G) depending on the type of toxin produced. Among them, clostridium perfringens type G is the main pathogenic bacterium of necrotic enteritis (Necrotic enteritis, NE) in birds.

Currently, the poultry industry is primarily concerned with the prophylactic treatment of necrotic enteritis in poultry by the use of antibiotics. However, more and more bacteria are reported to develop resistance to antibiotics, and thus there is an urgent need for a safe and effective method for preventing necrotic enteritis in birds.

Disclosure of Invention

Based on the above, the application provides a recombinant protein of clostridium perfringens G, vaccine and application, wherein the recombinant protein can effectively prevent and treat necrotic enteritis of birds.

According to one aspect of the present application, there is provided a recombinant protein of clostridium perfringens type G having the amino acid sequence shown in SEQ ID No. 3.

An isolated nucleic acid encoding the recombinant protein described above.

In one embodiment, the nucleic acid has a sequence as shown in SEQ ID NO.1 or SEQ ID NO. 2.

A recombinant vector comprising the nucleic acid described above.

A host cell having incorporated into its genome the nucleic acid described above or the recombinant vector described above.

In one embodiment, the host cell is a prokaryotic cell.

A vaccine comprising the recombinant protein, the nucleic acid or the recombinant vector.

In one embodiment, an adjuvant is also included.

Kit of parts comprising a vaccine as described above, and a container for the vaccination of said vaccine.

According to another aspect of the present application, there is provided the use of the recombinant protein, the nucleic acid or the recombinant vector described above for the preparation of a medicament for the prevention and treatment of clostridium perfringens type G infection.

Compared with the prior art, the method has the following beneficial effects:

the application develops recombinant proteins with the amino acid sequences shown as SEQ ID NO.3 aiming at alpha toxin and NetB toxin of clostridium perfringens G. The recombinant protein can generate effective immune protection to clostridium perfringens G, and has the characteristics of safety, stability, no toxic or side effect and the like.

In addition, the recombinant protein can be used in the form of a molecular vaccine or an oral vaccine, and is effective in preventing and treating necrotic enteritis of poultry caused by clostridium perfringens G.

Drawings

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 advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive effort to a person skilled in the art.

FIG. 1 is a map of a recombinant plasmid constructed in example 2 of the present application;

FIG. 2 is a gel electrophoresis chart of colony PCR identification in example 2 of the present application;

FIG. 3 is a graph showing the purification and identification results of recombinant proteins in example 2 of the present application;

FIG. 4 is a map of a recombinant plasmid constructed in example 3 of the present application;

FIG. 5 is a gel electrophoresis chart of colony PCR identification in example 3 of the present application;

FIG. 6 is a graph showing results of intestinal lesions of chicken in example 4 of the present application.

Detailed Description

The detailed description of the embodiments of the present application will be presented in order to make the above objects, features and advantages of the present application more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is, however, susceptible of embodiment in many other forms than those described herein and similar modifications can be made by those skilled in the art without departing from the spirit of the application, and therefore the application is not to be limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, and the like used in the present application are commercially available or may be prepared by existing methods.

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

Some embodiments of the present application provide a recombinant protein of clostridium perfringens type G having the amino acid sequence shown in SEQ ID No. 3.

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

MGSDPSVGNNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWEAAAKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGLE

the present application also provides an isolated nucleic acid encoding the recombinant protein described above.

The nucleic acid may be RNA or DNA.

In some of these embodiments, the nucleotide sequence of the above-described nucleic acid is codon optimized for a different host cell.

The codon optimization refers to optimizing a target gene by utilizing a preferred codon of a host cell under the condition of not changing an amino acid sequence, and improving the expression efficiency of the recombinant protein.

In some embodiments, the nucleic acid has a nucleotide sequence as set forth in SEQ ID NO.1 or SEQ ID NO. 2.

In addition, the amino acid sequence of the recombinant protein can be further matched with a sequence selected from SEQ ID NO:3, and a sequence substantially similar to the amino acid sequence of 3. The nucleotide sequence of the nucleic acid may also be identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 or SEQ ID NO. 2. By "substantially similar" is meant that a given nucleic acid or amino acid sequence shares at least 95% identity with a reference sequence, e.g., 96%, 97%, 98%, 98.5%, 99%, 99.5%. Alternatively, it is meant that a given nucleic acid or amino acid sequence has a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleic acid or amino acid difference from a reference sequence, which difference is preferably an amino acid substitution or deletion for a polypeptide. Preferably, substantially similar sequences also retain the unique activity of the polypeptide that is highly efficient in detecting endogenous antibodies. Substitutions are generally considered to be conservative substitutions, such as substitutions in aliphatic amino acids Ala, val, leu and Ile with each other, exchanges of hydroxyl residues Ser and Thr, exchanges of acidic residues Asp and Glu, exchanges between amide residues Asn and Gln, exchanges of basic residues Lys and Arg, and exchanges between aromatic residues Phe, tyr.

The "substantially similar" amino acid sequence may 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) as well as by chemical modification techniques, such as by the addition of one or more polyethylene glycol molecules, sugars, phosphates, and/or other such molecules, wherein one or more molecules are not naturally attached to the wild-type protein. Derivatives include salts. Such chemical modifications are described in detail in basic textbooks and in more detailed monographs, as well as in a number of research literature, and are well known to those skilled in the art. It will be appreciated that the same type of modification may be present at several sites in a given protein or polypeptide to the same or different extents. In addition, a given protein or polypeptide may contain many types of modifications. Modifications can occur anywhere in the protein or polypeptide, including the peptide backbone, amino acid side chains, and 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 phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking formation, cysteine formation, pyroglutamic acid formation, methylation, gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydrocarbylation and ADP-ribosylation, selenization, sulfation, transfer RNA-mediated addition of amino acids of proteins (such as arginylation), and ubiquitination. They may also bind to vitamins, such as biotin, folic acid, or vitamin B12.

The application also relates to a recombinant vector containing the nucleic acid.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). In some embodiments, the vectors described herein contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

In some embodiments, the vectors described herein may further comprise fragments of genes used in screening (e.g., antibiotic resistance genes), nucleic acids for generating fluorescent proteins, and the like. The fluorescent protein may be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.

The green fluorescent protein can adopt common GFP, and also can adopt modified GFP genes, such as enhanced GFP gene EGFP and the like; the blue fluorescent protein may be selected from EBFP, azuritc, tagBFP and the like; the yellow fluorescent protein may be selected from EYFP, ypct, phiYFP and the like; the orange fluorescent protein may be selected from mKO, mOrange, mBanana and the like; the red fluorescent protein may be selected from TagRFP, mRuby, mCherry, mKate and the like.

The present application also relates to a host cell, the genome of which is incorporated with the nucleic acid described above or the vector described above.

In some of these embodiments, the host cells of the present application are transformed with the vectors described above.

The term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to: prokaryotic cells such as E.coli, lactobacillus or Bacillus subtilis; fungal cells such as yeast cells or aspergillus, insect cells such as S2 drosophila cells or Sf9, 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), e.g., do not include fertilized eggs, embryos, germ stem cells, embryonic stem cells, and the like. Preferably, the host cell is a prokaryotic cell; more preferably E.coli or Lactobacillus.

The present application also relates to a vaccine comprising the recombinant polypeptide, the nucleic acid or the vector.

In some embodiments, the vaccine described above further comprises an adjuvant. Adjuvants suitable for use in the vaccines of the present application include adjuvants that enhance or amplify the immune response of antibodies, as well as adjuvants that enhance cell-mediated T cell epitope responses. Such adjuvants are well known in the art.

In some embodiments, the above-described adjuvant is selected from one or more of alum, complete Freund's adjuvant, incomplete Freund's adjuvant, squalene, squalane, muramyl dipeptide, MF59, AS03, monophosphoryl lipid A, flagellin, cpG-ODN, poly (I: C), and small molecules of aluminum or calcium salts. These adjuvants are well known in the art and are available from several commercial sources. Among them, 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 particular embodiments, the amount of the adjuvant is between about 2% (v/v) and 10% (v/v). In more specific embodiments, the amount of the adjuvant is between about 3% (v/v) and 6% (v/v).

In some embodiments, the vaccine described above further comprises a pharmaceutically acceptable carrier.

A "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 be, for example, sterile water or sterile physiological saline solution. In more complex forms, the carrier may be, for example, a buffer, which may contain further additives, such as stabilizers or preservatives. Aqueous or aqueous saline solutions and aqueous solutions of sugars (e.g., dextrose and/or glycerol) can be employed as carriers, particularly for injectable solutions. Furthermore, the carrier may be and/or comprise a hydrocolloid and/or a polymer solution, for example, to thicken an avian vaccine to be sprayed on an avian.

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

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

Further, the vaccine comprises an inactivated virus and/or an inactivated bacterium (e.g., vaccine) and/or an antigen of the vaccine. This may be derived from the microorganism pathogenic to avians in any suitable manner, for example as a "live" attenuated, inactivated or subunit antigen.

Typical immunization is achieved by Subcutaneous (SC) or Intramuscular (IM) injection, or the vaccine may also be administered via a transdermal patch, in a delayed release implant, scarification or topical application. Administration may also be via drinking water and/or food of the recipient bird.

The present application also relates to a kit of parts comprising a vaccine as described above, and a container for the vaccination of said vaccine.

The application also relates to a preparation method of the vaccine, which comprises the following steps:

culturing the above host cells under proper conditions, collecting culture solution and/or lysate of host cells, and separating and purifying to obtain the vaccine.

The application also relates to application of the recombinant protein, the nucleic acid or the recombinant vector in preparation of medicines for preventing and treating G clostridium perfringens infection.

The present application further provides a method of protecting an avian from clostridium perfringens type G infection comprising administering to an avian animal a prophylactically effective amount/therapeutically effective amount of a vaccine of the present application.

The term "avian" refers to wild or domesticated birds, particularly chickens, ducks, geese, swans, geese, pigeons, quails and the like.

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

The present application will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the present application.

Example 1: screening of coding genes

The amino acid sequence shown as SEQ ID NO.3 is obtained by screening aiming at alpha toxin and NetB toxin of clostridium perfringens G.

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

and (3) aiming at different host cells, carrying out codon optimization to respectively obtain coding gene sequences with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.

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

CATATGGGCAGCGATCCGAGCGTGGGTAATAATGTGAAAGAACTGGTTGCCTATATTAGCACCAGCGGTGAAAAAGATGCAGGTACCGATGATTATATGTATTTTGGTATTAAGACCAAGGACGGTAAAACCCAGGAATGGGAAATGGATAATCCGGGTAATGATTTTATGGCCGGTAGCAAAGATACCTATACCTTTAAACTGAAAGACGAAAATCTGAAAATCGATGATATCCAGAATATGTGGATTCGCAAACGCAAATATACCGCCTTTCCGGATGCCTATAAACCGGAAAATATTAAAGTGATTGCGAATGGTAAGGTTGTGGTTGATAAAGATATTAATGAGTGGGAAGCCGCCGCAAAATATTATGGTAAAATGAAATGGCCGGAAACCTATCGTATTAATGTTAAAAGTGCAGATGTGAATAACAACATTAAGATTGCAAACAGTATCCCGAAAAATACCATTGATAAAAAGGATGTTAGCAACAGCATTGGTTATAGTATTGGCGGTAATATTAGCGTGGAAGGTAAAACCGCAGGTGCCGGTATTAATGCAAGCTATAATGTTCAGAATACCATTAGTTACGAGCAGCCGGATTTTCGTACCATTCAGCGCAAAGATGATGCAAATCTGGCCAGTTGGGATATTAAATTTGTGGAAACCAAAGATGGTTACAATATTGATAGCTATCACGCCATTTATGGCAATCAGCTGTTTATGAAAAGCCGTCTGTATAATAATGGTCTCGAG

the nucleotide sequence shown in SEQ ID NO.2 is:

CCATGGGAATTCAGATCTTATGCTTTTGTTATAAGTTAGCACAAAAAAGCAGAAAATAAAAAGTAGAAATAAAAAAAGATGTTTTTTTGCCCATATCTCTATGAAAAAAACTGTGAAATGTGTAAAATATGGATGAAACATTGAATTTAAAAGGAGATATTTCATGAAGAAGGAATTGTCATTCCATGAAAAATTGTTGAAG

TTGACTAAGCAACAAAAGAAAAAGACTAACAAGCATGTTTTCATTGCTATT

CCAATTGTTTTCGTTTTGATGTTTGCTTTTATGTGGGCTGGTAAAGCTGAAA

CTCCAAAAGTTAAGACTTATTCAGATGATGTTTTGTCAGCTTCATTTGTTGG

TGATATTATGATGGGTCGTTATGTTGAAAAGGTTACTGAACAAAAAGGTGC

TGATTCAATTTTTCAATACGTTGAACCAATTTTCCGTGCTTCAGATTATGTTG

CTGGTAATTTTGAAAACCCAGTTACTTATCAAAAGAACTATAAGCAAGCTG

ATAAGGAAATTCATTTGCAAACTAATAAGGAAAGCGTTAAGGTTTTAAAGG

ATATGAATTTCACTGTTTTGAACTCAGCTAATAACCATGCTATGGATTATGGT

GTTCAAGGTATGAAGGATACTTTAGGTGAATTTGCTAAGCAAAATTTGGATA

TTGTTGGTGCTGGTTATTCATTATCAGATGCTAAAAAAAAGATTAGCTACCA

AAAGGTTAACGGTGTTACTATTGCTACTTTAGGTTTTACTGATGTTTCAGGT

AAGGGTTTTGCTGCTAAGAAGAATACTCCAGGTGTTTTACCAGCTGATCCA

GAAATTTTTATTCCAATGATTAGCGAAGCTAAGAAACATGCTGATATTGTTG

TTGTTCAATCACATTGGGGTCAAGAATATGATAATGATCCAAATGATCGTCA

ACGTCAATTAGCTCGTGCTATGTCAGATGCTGGTGCTGATATTATTGTTGGT

CATCATCCACATGTTTTAGAACCAATTGAAGTTTATAACGGTACTGTTATTTT

CTACTCATTGGGTAATTTCGTTTTCGATCAAGGTTGGACTCGTACTCGTGAT

TCAGCTTTAGTTCAATATCATTTAAAGAAGAACGGTACTGGTCGTTTTGAA

GTTACTCCAATTGATATTCATGAAGCTACTCCAGCTCCAGTTAAGAAGGATT

CATTAAAGCAAAAGACTATTATTCGTGAATTGACTAAGGATTCAAACTTTG

CTTGGAAAGTTGAAGATGGTAAGTTAACTTTTGACATTGATCATTCAGACA

AGTTAAAGTCAAAGGAAGGTAAGTCATCAGGTTCAGGTTCAGAATCAAAA

TCAACTGGATCCGCTCCACCACATGCTTTATCAGGTTCAGATCCATCAGTTG

GTAATAATGTTAAGGAATTAGTTGCTTACATTAGCACTTCAGGTGAAAAAG

ATGCTGGTACTGATGATTATATGTATTTTGGTATTAAGACTAAGGACGGTAA

AACTCAAGAATGGGAAATGGATAATCCAGGTAATGATTTTATGGCTGGTTC

AAAAGATACTTATACTTTTAAGTTGAAGGACGAAAACTTGAAAATTGATGA

TATTCAAAACATGTGGATTCGTAAACGTAAATATACTGCTTTTCCAGATGCT

TATAAGCCAGAAAATATTAAAGTTATTGCTAACGGTAAGGTTGTTGTTGATA

AAGATATTAACGAATGGGAAGCTGCTGCTAAATATTATGGTAAAATGAAGT

GGCCAGAAACTTATCGTATTAATGTTAAGTCAGCTGATGTTAATAACAACAT

TAAGATTGCTAACAGCATTCCAAAAAACACTATTGATAAGAAGGACGTTTC

AAATTCAATTGGTTATTCAATTGGTGGTAACATTTCAGTTGAAGGTAAAACT

GCTGGTGCTGGTATTAATGCTTCATATAATGTTCAAAACACTATTAGCTACG

AACAACCAGATTTTCGTACTATTCAACGTAAAGATGATGCTAATTTGGCTTC

ATGGGATATTAAGTTTGTTGAAACTAAGGATGGTTACAATATTGATTCATAC

CATGCTATTTACGGTAATCAATTATTCATGAAGAGCCGTTTATACAATAACG

GTGGATCCGAGCTCGGGCCCTCTAGAGTCGACCTCGAGCACCACCACCAC

CACCACTGATAACCCGGGTAATCTGAAGAAAAAGGAGGCTAGTATACTAGC

CTCCTTTTTCTTCAGATTACCCGGGGCGGCCGCAAGCTT

example 2: preparation of recombinant proteins

(1) The gene fragment with the nucleotide sequence shown as SEQ ID NO.1 obtained by manual screening combination is subjected to total gene synthesis, ndeI and XhoI restriction enzyme double-enzyme digestion are carried out simultaneously with a prokaryotic expression vector pET-30a (+), and then the nucleic acid fragments are respectively recovered and connected with T4 DNA ligase overnight; the recombinant plasmid map after ligation is shown in FIG. 1.

(2) The ligation products were heat-shock transformed into BL21 (DE 3) infected cells, and antibiotic pressure screening was performed using LB plates containing 50. Mu.g/ml kanamycin, and recombinant bacteria were obtained after colony PCR identification (as shown in FIG. 2) and nucleic acid sequencing analysis. The bacterium contains the recombinant plasmid constructed in the step (1).

(3) Selecting single colony of the recombinant bacteria selected in the step (2) to 5mL of LB liquid medium containing 50 mug/mL kanamycin, culturing overnight, inoculating to 100mL of LB liquid medium containing 50 mug/mL kanamycin according to the inoculum size of 1%, culturing to logarithmic phase at 25 ℃, adding 1mM IPTG inducer, culturing for 16 hours, and collecting the bacterial cells. PBS buffer was added to the cells, followed by washing twice successively, PBS buffer containing 10mL of 1% Triton X-100 was added, the cells were sonicated on ice, and centrifuged at 12000rpm for 20min, and the supernatant Q1 was collected. And (3) separating and purifying target protein in supernatant Q1 by using a Ni-Sepharose affinity chromatographic column, and collecting eluent to obtain recombinant protein of clostridium perfringens G.

The result shows that the recombinant bacteria can effectively express the target protein through induction, the size of the target protein is about 35Kda, and the purification result of the recombinant protein is shown in figure 3.

Example 3: preparation of oral vaccine

(1) Artificially synthesizing a codon optimized gene fragment (the nucleotide sequence is shown as SEQ ID NO. 2), cloning by a primer with a Not I restriction site primer (the nucleotide sequence is shown as SEQ ID NO. 4) and a HindIII restriction site primer (the nucleotide sequence is shown as SEQ ID NO. 5) to obtain a fragment 1, cloning by a primer with a BamH I restriction site (the nucleotide sequences are shown as SEQ ID NO.6 and SEQ ID NO. 7) to obtain a fragment 2, sequentially performing enzyme digestion on the two fragments to be connected to a pNZ8148 vector, and screening correct positive clones by colony PCR to obtain a plasmid 8148-CACN, wherein the plasmid map is shown as figure 4.

TABLE 1 primer information

(2) NZ9000 competent cells were prepared. Firstly, streaking NZ9000 bacterial liquid on a GM17 solid culture medium, and carrying out stationary culture for 24-36 h at 30 ℃ under anaerobic conditions; picking single colonies after plate streaking into 5mL GM17 liquid culture medium, and carrying out stationary culture at 30 ℃ for 12-16 h under anaerobic conditions; inoculating into 50mL fresh G/L-SGM17 liquid culture medium at 5% ratio, and culturing at 30deg.C under anaerobic condition until OD ₆₀₀ Reaching 0.3 to 0.4 (estimated 2 to 3 hours); centrifuging at 4deg.C for 10min at 5000g, discarding supernatant, and retaining thallus precipitate; adding 5mL of washing solution III into the bacterial precipitate, re-suspending the bacterial precipitate, and standing for 30min at room temperature; centrifuging at 4deg.C for 10min at 5000g, discarding supernatant, and retaining thallus precipitate; adding 5mL of washing liquid I (precooling) into the bacterial precipitate, and re-suspending the bacterial precipitate; centrifuging at 4deg.C for 10min at 5000g, discarding supernatant, and retaining thallus precipitate; adding 5mL of washing liquid II (precooling) into the bacterial precipitate, and re-suspending the bacterial precipitate; centrifuging at 4deg.C for 10min at 5000g, discarding supernatant, and retaining thallus precipitate; 1mL (1/50 culture volume) of washing solution I (precooling) is added to the bacterial cell sediment, bacterial cells are resuspended, competent cells (ice bath) are packed according to 100 mu L/tube, and finally the bacterial cells are stored in a refrigerator at-80 ℃ for standby.

Wherein the washing liquid I comprises 0.5mol/L sucrose and 10% glycerol; washing solution II comprises 0.5mol/L sucrose, 10% glycerol and 50mmol/L EDTA; washing III included 100mmol/L lithium acetate dihydrate, 10mmol/L Dithiothreitol (DTT), 0.6mol/L sucrose and 1mol/L Tris-HCl (pH 7.5).

(3) 400ng of plasmid 8148-CACN prepared in the step (1) is added into 100 mu L of NZ9000 competent cells, and the mixture is placed on ice for 5min after uniform mixing; rapidly transferring to a 2mm electric shock cup (precooled for more than 10 min), placing in an electric converter, and discharging for 4.5 ms-5 ms according to 2.5 kV; after electrotransformation, 900 mu L of recovery culture medium (precooling) is quickly added, and ice bath is carried out for 5min; transferring the electrotransfer product to a 1.5mL centrifuge tube, and standing and culturing for 2h at 30 ℃; taking 100 mu L of GM17 solid culture medium coated with 1/2000 and 1/4000 chloramphenicol antibiotics, and standing and culturing for 36-48 h at 30 ℃ under anaerobic condition; picking the transformant for colony PCR identification, and obtaining a colony consistent with the expected size as shown in the result of FIG. 5; thus preparing an oral vaccine.

In the embodiment, a nucleotide sequence for encoding the recombinant protein of clostridium perfringens G is connected with an expression vector, and is positioned on the cell surface of lactobacillus through pgsA (poly-cobalamine synthetase A) for display expression; thus preparing an oral vaccine.

Example 4: animal experimental evaluation of clostridium perfringens vaccine G

(1) 817 broilers, which were not vaccinated at 1 day of age, were selected and divided into 3 groups of 30 experimental chickens. All test chickens were kept in cages without coccidian environment, were allowed to drink and feed freely. Wherein the immunization groups were subcutaneously injected with the recombinant protein prepared in example 2 at 1 day of age, 7 days of age and 14 days of age, respectively. Thereafter, the immunization group and the positive control group were subjected to attack treatment of clostridium perfringens type G at the age of 26 to 29 days according to 8X 10 ⁸ CFU/proceed only.

Weighing at 1 day of age, 21 days of age and 30 days of age respectively, and calculating average weight gain and relative weight gain rate; at 30 days of age, survival rate was calculated; the results are shown in Table 2.

Survival = survival rate of 30 days old surviving chickens/21 days old surviving chickens 100%

Average weight gain (g) =30-21-day-old average body weight (g)

Relative weight gain (%) = (average weight gain of infected chickens/average weight gain of uninfected chickens) ×100%

TABLE 2

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

(2) Part of chickens were dissected and collected for the duodenum, jejunum, ileum and cecum, and lesion scores and photographic recordings were performed. The results are shown in Table 3 and FIG. 6.

The scoring criteria were as follows: 0 = no obvious lesions, 1 = congestion of intestinal mucosa, 2 = focal necrosis or ulcers (1-5 lesions), 3 = focal necrosis or ulcers (6-15 lesions); 4 = focal necrosis or ulceration (16 or more lesions)

TABLE 3 Table 3

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

Therefore, after the recombinant protein is used for immunizing chickens, intestinal lesions are obviously improved compared with a positive control group, and the relative weight gain rate and the survival rate are similar to those of a negative control group, so that the recombinant protein can effectively improve the intestinal damage caused by clostridium perfringens G.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims

1. A recombinant protein of clostridium perfringens type G, characterized in that it has an amino acid sequence shown in SEQ ID No. 3.

2. An isolated nucleic acid encoding the recombinant protein of claim 1.

3. The nucleic acid according to claim 2, wherein the sequence of the nucleic acid is shown as SEQ ID No.1 or SEQ ID No. 2.

4. A recombinant vector comprising the nucleic acid according to any one of claims 2 to 3.

5. A host cell comprising the nucleic acid of any one of claims 2 to 3 or the recombinant vector of claim 4.

6. The host cell of claim 5, wherein the host cell is a prokaryotic cell.

7. A vaccine comprising the recombinant protein of claim 1, the nucleic acid of any one of claims 2 to 3, or the recombinant vector of claim 4.

8. The vaccine of claim 7, further comprising an adjuvant.

9. Kit of parts, characterized in that it comprises a vaccine according to any one of claims 7 to 8, and a container for the vaccination of said vaccine.

10. Use of the recombinant protein of claim 1, the nucleic acid of any one of claims 2 to 3 or the recombinant vector of claim 4 for the preparation of a medicament for the prevention and treatment of clostridium perfringens G infection.