CN110157655B

CN110157655B - Strain for non-toxic clostridium emphysema genetic engineering subunit vaccine and application thereof

Info

Publication number: CN110157655B
Application number: CN201910426739.1A
Authority: CN
Inventors: 杜吉革; 陈小云; 刘莹; 彭小兵; 薛麒; 朱真; 李启红; 印春生; 姚文生; 康凯
Original assignee: China Institute of Veterinary Drug Control
Current assignee: China Institute of Veterinary Drug Control
Priority date: 2019-05-22
Filing date: 2019-05-22
Publication date: 2021-06-01
Anticipated expiration: 2039-05-22
Also published as: CN110157655A

Abstract

The invention relates to a nontoxic emphysema clostridium genetic engineering subunit vaccine and application thereof, the strain for the nontoxic emphysema clostridium genetic engineering subunit vaccine prepared by the invention is escherichia coli which recombines and expresses codon-optimized clostridium emphysema flagellin and cytotoxin A recombinant fusion protein, and the clostridium emphysema genetic engineering subunit vaccine produced by the strain can ensure the safety of the vaccine to the maximum extent and keep the effectiveness thereof. Meanwhile, the vaccine antigen expressed by the strain can be expressed in a soluble form, so that the influence of a complicated process of inclusion body denaturation and renaturation on the immunogenicity of the antigen protein is avoided, and the preparation time and the production cost of the target protein are reduced; on one hand, the antigen of the fusion protein can be improved, so the vaccine has the advantages of simple preparation process, low immune dose, good immune efficacy and the like, and is an ideal candidate vaccine for upgrading and updating the current clostridium emphysema gangreniformis vaccine in China.

Description

Strain for non-toxic clostridium emphysema genetic engineering subunit vaccine and application thereof

Technical Field

The invention relates to a strain for a non-toxic clostridium emphysema genetic engineering subunit vaccine and application thereof. Belongs to the field of biological products for animals.

Background

Clostridium emphysema, also commonly known as black leg disease, primarily infects ruminants, especially grazing newborn ruminants, which are most sensitive to clostridium emphysema. The disease has short course and acute onset, and the patient usually dies without treatment. Therefore, vaccination is the main means for preventing and controlling the disease, and the commercial Gaoquan vaccine is mainly an inactivated vaccine of two strains of virulent strains C54-1 and C54-2. Although the vaccine has certain effect on preventing the emphysema and the cellulitis of the animals, the vaccine still has some defects in the using process, for example, the vaccine immunity is easy to cause local inflammation and toxic reaction of the animals; the preparation process relates to the inactivation of clostridium aeroma with strong toxicity, and has the biological safety hidden troubles of strain leakage or incomplete inactivation and the like. Therefore, the development of the gene engineering vaccine of the clostridium emphysema gangrensis with good safety, high effective antigen content and strong immunogenicity is the development direction in the future.

The major virulence factors of C.aeroma are flagella, cytotoxin A (Ccta), and sialidase, as well as other virulence factors, including those representing aerotolerant hemolysins, deoxyribonuclease, hyaluronidase, and non-aerotolerant hemolysins (Vilei EM, Johansson A, Schlater Y, Redhead K, free J.genetic and functional characterization of the NanA sialidase from Clostridium chauvoei.vet Res 2011; 42 (1: 2)). Among them, flagella are closely related to the virulence of emphysema gangrene, and flagellin has good immunogenicity and is capable of providing an immunoprotective response to emphysema gangrene (Chandler HM, Gulasekharam J. the protective antigen of a highlym immunogenic strain J. Gen Microbiol 1974; 84(1) 128-34; Tamuray, Kijima-Tanaka M, Aokia, Ogilub Y, Takahashi T. reversible expression of biological and fluidic antigen of microorganism and microorganism, 141(Pt 3): 605. 610). The existing research finds that the exogenously expressed flagellin FliA (C) can induce mice to generate better humoral immune response and cellular immune response (emphysema and gangrene nanA and fliA fusion gene subunit vaccine preparation and immune effect evaluation [ D]Yanbian university, 2014.). In 2012, Joachim et al discovered a novel toxin of clostridium emphysema, named clostridium emphysema gangrensis cytotoxin a (ccta). The toxin is one of the leukocidin superfamily of bacterial toxins, the family of β -pore forming toxins, and is well conserved among clostridium pneumonectum. Further research shows that both natural Ccta and fusion protein rCcta expressed by Escherichia coli have strong cytotoxicity and hemolytic activity. Rabbit polyclonal antibodies against rCctA were able to completely neutralize the cytotoxic and hemolytic activities of rCctA and aeroma cultures, indicating that CctA is the major cytotoxic and hemolytic factor of clostridium aerocremastadens. In addition, Joachim et al have also discovered that animals immunized with LTB can achieve control of emphysema using an exogenous fusion protein rCctAImmunoprotection of Clostridium (Frey J, Johansson A, sbyle Bu rki, et al cytotoxin Ccta, a major viral factor of Clostridium chauvoei transducing protective immunological infection bacteria [ J]Vaccine,2012,30(37), 5500-. Recent studies have found that the middle region of Ccta (amino acids 95 to 261, hereinafter referred to as Ccta)_N) Can be expressed in eukaryotic cells, can be used as a nucleic acid vaccine for immunization, induces experimental animals to generate better humoral immune response and cellular immune response (Qiqiang cattle emphysema gangrene Ccta gene nucleic acid vaccine preparation and immune effect evaluation [ D ]].2017.)。

Disclosure of Invention

The invention aims to prepare a nontoxic recombinant fusion protein (rFliACCTA) by using constructed escherichia coli expressing recombinant fusion protein of clostridium pneumonectum flagellin FliA (C) and nontoxic fragments (amino acids from 95 th to 261 th) of cytotoxin A (CctA)_N) And can be used for preventing diseases caused by infection of Clostridium pneumonectatum.

Technical scheme of the invention

1. A non-toxic bacillus pyometra gene engineering subunit vaccine strain and its production method and application are characterized by that the said strain is a recombinant fusion protein (rFliACCTA) of recombinant expression clostridium pyometra flagellin (FliA (C)) and cytotoxin A (CctA)_N) The Escherichia coli (Escherichia coli) BL21(DE3) cell of (1), which is named as Escherichia coli BL/MA strain and has been delivered to the common microbiology center of the china institute of science, china institute of microbiology,

west way

1, 3, north chen, north west way, beijing, on 2019, 03 and 08 days, with the accession number: CGMCC No. 17319.

The strain can be used for producing clostridium emphysema and flagellin and cytotoxin A recombinant fusion protein, so that a clostridium emphysema genetic engineering subunit vaccine is prepared, and the vaccine is used for preventing diseases caused by clostridium emphysema infection.

2. The invention relates to a strain for a non-toxic clostridium emphysema genetic engineering subunit vaccine and application thereof, which are characterized in that rFliACCTA expressed by the strain_NContains flagellin FliA (C) and cytotoxin A (CctA); all the other Chinese medicinal herbsBiotoxin a (ccta) contains only a non-toxic fragment (amino acids 95-261);

the recombinant fusion protein rFliACCta_NAnd the vaccine is avirulent, so that the biological safety risk in the vaccine production process is greatly reduced.

3. The invention relates to a strain for a non-toxic clostridium emphysema genetic engineering subunit vaccine and application thereof, which are characterized in that a fusion protein rFliACCtA expressed by the strain_NThe coded gene sequence is optimized by a codon, and high expression and soluble expression can be realized in escherichia coli more easily.

4. The invention relates to a strain for a non-toxic clostridium emphysema genetic engineering subunit vaccine and application thereof, which are characterized in that a fusion protein rFliACCtA expressed by the strain_NThe C-terminus contains a tag 6 histidine (6 × His) tag to facilitate protein purification.

5. The invention relates to a nontoxic clostridium emphysema genetic engineering subunit vaccine strain and application thereof, which is characterized in that the preparation method of the clostridium emphysema genetic engineering subunit vaccine is as follows: using said expression rFliACCta_NThe Escherichia coli BL/MA strain is used as a vaccine production strain, and is prepared by fermentation culture, induction expression, thallus crushing, soluble antigen protein separation and purification, and then adding an adjuvant and mixing.

6. The invention claim 1 of the invention relates to a nontoxic emphysema gangrene clostridium genetic engineering subunit vaccine strain and application thereof, which is characterized in that the strain is used for expressing fusion protein rFliACCTA_NThe "host cell" of (a) encompasses prokaryotic and/or eukaryotic cells, which may be a Clostridium sporogenes, Clostridium perfringens, Clostridium acetobutylicum, Bacillus cereus, Bacillus thuringiensis, Bacillus caldolyticus, Bacillus stearothermophilus, Bacillus anthracis, Bacillus megaterium, Bacillus subtilis, Escherichia coli or yeast cells;

the host cell is preferably an E.coli host cell, in particular E.coli BL21(DE3) or Rosetta (DE 3).

The invention has the beneficial effects

The invention relates to clostridium tetani flagellin of emphysemaFliA (C) and a nontoxic fragment (amino acids from 95 th to 261 th) of CctA are subjected to fusion expression to obtain a recombinant fusion protein (rFliACCTA) which is nontoxic to animal bodies_N). The invention further discloses a composition containing the rFliACCTA_NExpression vectors and host cells encoding the genes. rFliACCta of the invention_NIs completely non-toxic in mice and exhibits good immunogenicity and immunoprotection in guinea pig models. rFliACCta of the invention_NOr the coding gene can be applied to the preparation of the genetic engineering subunit vaccine for preventing the infection of the clostridium emphysema. The strain for the non-toxic clostridium emphysema genetic engineering subunit vaccine can be used as an ideal strain for preparing the clostridium emphysema genetic engineering subunit vaccine, and compared with the current commercialized clostridium emphysema inactivated vaccine in China, the strain greatly reduces the biological safety risk in the vaccine production process. Meanwhile, the method for producing the fusion protein by using the strain has good stability and short time consumption, and the guinea pig immunized by the subunit vaccine prepared by using the fusion protein can resist the attack of the virulent bacterial liquid of the lethal clostridium gangreniformis. In addition, by virtue of the advantage of high concentration of the recombinant fusion protein, when combined seedlings are prepared together with other antigens, the combined seedlings can be prepared without increasing the using dosage of the combined seedlings, so that the development of the combined seedlings is greatly facilitated.

In view of the fact that the existing commercial clostridium emphysema inactivated vaccine in China needs to be inactivated and detoxified by formaldehyde, the existing commercial clostridium emphysema inactivated vaccine has potential biological safety hazards and also influences the safety of the vaccine in field use; meanwhile, the time and culture medium cost of the current commercial vaccine in the production process are too high, so that the vaccine efficacy is unstable. Therefore, the strain for the non-toxic clostridium emphysema gene engineering subunit vaccine is an ideal candidate strain for upgrading and updating the current clostridium emphysema clostridium perfoliatum inactivated vaccine in China.

In conclusion, it can be seen that: (1) the invention firstly fuses and expresses the flagellin FliA (C) and Ccta of clostridium emphysema and realizes the soluble expression in escherichia coli, thereby avoiding the influence of the complicated process of the denaturation and renaturation of the inclusion body on the immunogenicity of the antigen protein and reducing the preparation time and the production cost of the target protein. (2) Selection of Ccta avirulent fragment(amino acids 95 to 261) to obtain a non-toxic recombinant fusion protein. (3) For coding recombinant fusion protein rFliACCta_NThe gene sequence of (A) is subjected to codon optimization, and high-efficiency expression and soluble expression in escherichia coli are realized. (4) Recombinant expression of rFliACCta_NThe rFliACCta prepared by taking Escherichia coli BL21(DE3) cells as strains for clostridium emphysema genetic engineering subunit vaccine_NCan be used as subunit vaccine to replace the traditional method for culturing the pathogenic clostridium pneumonectum, thereby greatly reducing the biosafety risk in the production process.

The invention relates to biomaterial resource information

The microorganism related to the invention is: BL21(DE3) cell strain of Escherichia coli (Escherichia coli) expressing recombinant fusion protein of Clostridium emphysema and flagellin FliA (C) and CctA nontoxic fragment (amino acids 95-261) is named as Escherichia coli BL/MA strain, which has been deposited by the China Committee for culture Collection of microorganisms of institute of microbiology, China institute of sciences, Ministry of microbiology, No.1, North Cheng, south China, province, Youngia, 3, in 2019, 03 and 08 days, and has the following deposition numbers: CGMCC No. 17319.

Drawings

FIG. 1: rFliACCta_NSDS-PAGE identification of expression

M₁Protein marker; BSA (1. mu.g); BSA (2. mu.g); 3, empty vector cell lysate; cell lysate induced at 4:15 ℃ for 16 h; 5: cell lysate induced at 37 ℃ for 4 h; 6, supernatant of the cell lysate of the empty vector; 7, inducing cell lysate supernatant for 16h at 15 ℃; cell lysis supernatant induced at 37 ℃ for 4 h; 9, precipitating the cell lysate of the empty carrier; precipitating the cell lysate induced at 10:15 ℃ for 16 h; 11, cell lysis precipitation induced at 37 ℃ for 4 h;

FIG. 2: rFliACCta_NExpressed Western blot (with anti-His antibody) identification results

M2 Western blot marker; 1, empty vector cell lysate; cell lysate induced at 2:15 ℃ for 16 h; 3, cell lysate induced at 37 ℃ for 4 h; cell lysate supernatant induced at 4:15 ℃ for 16 h; 5, inducing cell lysis supernatant at 37 ℃ for 4 h; 6, 15 ℃ and 16h induced cell lysate precipitation; 7, cell lysis precipitation induced at 37 ℃ for 4 h;

FIG. 3: m1, Protein marker; cell lysis precipitation induced at 1:15 ℃ for 16 h; cell lysis supernatant induced at 2:15 ℃ for 16 h; 3, flowing through liquid; 4, washing liquid; 5:20mM imidazole eluent; 6:50mM imidazole eluent; 7: 300mM imidazole eluent

Detailed description of the invention

1. The clostridium emphysema of the invention flagellin FliA (C) and cytotoxin A (CctA) recombinant fusion protein (rFliACCtA)_N) Wherein:

(1) the term "FliA" means Clostridium pneumonectans flagellin FliA (C), "Ccta_N"means a non-toxic fragment (amino acids 95 to 261) containing the cytotoxin A (CctA). "FliA" and "Ccta_N"also encompasses one or more modified (including chemically and genetically modified) nontoxic fragments of flia (c) and CctA. The term "genetically modified" means that one or more amino acid bases are deleted, substituted or added.

The methods of the invention may be used to generate homologues of any FliA (C) and Ccta or derivatives thereof, including for example polypeptides of the amino acid sequence of FliA (C) SEQ ID No.2(GenBank accession AHC53287.1) and Ccta SEQ ID No.4(GenBank accession AFN27591.1) and derivatives thereof. The term "derivative" encompasses amino acid mutations such as additions, substitutions, deletions or truncations of one or more amino acid residues.

Sequence 2

Sequence 4

rFliACctA_NMay comprise polypeptide sequences which are homologues or derivatives having at least 50% sequence identity with the flia (c) and CctA sequences referred to above.

rFliACctA_NMay comprise a derivative of any one of SEQ ID No.2 and SEQ ID No.4 or a derivative of one of the polypeptide sequences corresponding to the accession numbers identified herein. Wherein the derivative has one or more point mutations and/or one or more additional amino acid residues. In another aspect, the recombinant fusion protein can comprise a polypeptide sequence having at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to one of the sequences of SEQ ID No.1 and SEQ ID No.2 or to a sequence of polypeptide sequences corresponding to an accession number identified herein.

(2) The term "sequence identity" refers to determining identity between a reference amino acid sequence and a query sequence, wherein the sequences are aligned such that the highest level of matching is achieved, and the sequence identity can be calculated using published techniques or methods coded in computer programs (e.g., BLASTP, BLASTN, FASTA). Percent identity values can be calculated over the entire amino acid sequence or over a region of the amino acid sequence.

(3)rFliACctA_NAdditional amino acid residues may be contained at the N-terminus or C-terminus or at internal positions. The additional amino acid residues may be flanked by one or more protease cleavage sites, which may function as a detectable tag and or allow binding to a solid support, an example being a His-tag.

(4) Encoding the rFliACCta_NThe nucleic acid molecule of (a) may optionally comprise regulatory elements according to the present patent. The term "regulatory element" as used herein refers to regulatory elements of gene expression, including transcription and translation, and includes elements such as TATA boxes, promoters, enhancers, ribosome binding sites, and the like. The regulatory element may comprise one or more homologous regulatory elements and/or heterologous regulatory elements. A "homologous regulatory element" is a regulatory element involved in the gene expression of a nucleic acid molecule or polypeptide in said wild-type cell. A "heterologous regulatory element" is a regulatory element which is not involved in the gene expression of a nucleic acid molecule or polypeptide in said wild-type cell. In addition, regulatory elements for inducible expression, such as inducible promoters, may also be used.

(5) Encoding the rFliACCta_NThe nucleic acid molecule of (a) can be designed to facilitate high levels of expression in a host cell, particularly a bacterial host cell, preferably an E.coli cell, e.g., codon optimization according to an E.coli expression system, etc. In another aspect, the invention can be used arbitrarily, including encoding the rfiiaccta_NThe expression vector of (a) the nucleic acid molecule, the vector may be suitable for expressing the recombinant fusion protein in vitro and/or in vivo. The vector may be a vector for transient and/or stable gene expression, may additionally comprise regulatory elements and/or selectable markers, and may be of viral, phage or bacterial origin. For example, the expression vector may be the pET30a vector.

(6) Encoding the rFliACCta_NThe nucleic acid molecule or expression vector of (a) may be expressed by a host cell. As used herein, the term "host cell" encompasses prokaryotic and/or eukaryotic cells suitable for translation of the nucleic acid molecule or the vector. Such host cells include not only host cells that do not express flia (c) and/CctA or homologues thereof, but also host cells that express both toxins or homologues, such as wild-type. The term "host cell" as used herein encompasses cells which may be clostridium sporogenes, clostridium perfringens, clostridium acetobutylicum, bacillus cereus, bacillus thuringiensis, bacillus caldolyticus, bacillus thermophilus, bacillus anthracis, bacillus megaterium, bacillus subtilis, escherichia coli or yeast cells. Preferably, the host cell is an E.coli host cell, in particular E.coli BL21(DE3) or Rosetta (DE 3). The Escherichia coli BL21(DE3) expressing FliA (C) and CctA recombinant fusion protein is named as BL/MA strain.

2. Construction, expression and identification of Escherichia coli BL/MA strain

(1) Gene synthesis

According to the natural gene sequences of FliA (C) (sequence 1) and Ccta (sequence 3), after codon optimization, the coding gene containing FliA (C) and the coding gene containing the middle region (amino acids 95-261) of Ccta are expressed in series, thus obtaining the recombinant fusion protein which is nontoxic to animal bodies. Meanwhile, an amino acid tag coding sequence used for purification is added at the C end of the recombinant fusion protein. The gene sequence is synthesized by a chemical synthesis method.

(2) Construction of recombinant fusion protein expression vectors

The artificial synthesized gene is used as a template, a designed primer pair is adopted to carry out PCR amplification to obtain a target DNA strip, and after recovery, the target DNA strip is connected with a prokaryotic expression vector after double enzyme digestion is carried out simultaneously, so that the prokaryotic expression vector inserted with the target gene is obtained. The ligated plasmid was transformed into DH 5. alpha. competent cells, and a single clone was picked up into LB liquid medium containing kanamycin, and cultured overnight with shaking at 37 ℃ to extract the plasmid for use.

(3) Construction of genetically engineered strains expressing recombinant fusion proteins

Transforming the plasmid into competent cells of Escherichia coli BL21(DE3), selecting a single clone to LB liquid culture medium containing kanamycin, carrying out shake culture at 37 ℃ overnight, carrying out PCR identification to obtain a positive strain after the target DNA fragment is contained, and adding 50% of glycerol LB with the same volume and freezing at-70 ℃.

(4) Expression and characterization of recombinant fusion proteins

Escherichia coli (E.coli) BL/MA strain was inoculated into 4mL of LB liquid medium containing kanamycin, cultured with shaking, and when OD is reached₆₀₀When the concentration is 0.6-0.8, IPTG solution with the final concentration of 0.5mMM is added and the mixture is respectively placed at 37 ℃ and 15 ℃ for induced culture for 4h and 16 h. After the bacterial liquid culture is finished, the thalli are centrifugally collected, and 10ml of lysis solution (0.02 mol/LTris buffer solution (pH value 7.2) and 0.3mol/LNaCl are added according to the body temperature of each gram of thalli]Resuspending the thallus according to the proportion, and carrying out ultrasonic disruption on the thallus in an ice water bath for 30min under the conditions: the operation time is 9s, the pause time is 9s, and the ultrasonic power is 400W. The crushed bacterial liquid is treated at 4 ℃ at 12000r/minCentrifuging for 10min, and collecting supernatant. And (3) adding 10 mul of 4 xSDS-PAGE loading buffer solution into 30 mul of supernatant, acting for 10min at 70 ℃, performing 12% SDS-PAGE electrophoresis, and performing Western blot identification on the supernatant by using an anti-His antibody to finally determine the optimal conditions of expression.

4. Purification of recombinant fusion proteins

Escherichia coli BL/MA strain was inoculated into 1L LB liquid medium containing kanamycin for fermentation culture, followed by shaking culture at 37 ℃ for OD₆₀₀And (3) when the expression level is 0.6-0.8, performing induced expression according to the determined optimal expression conditions, collecting bacteria, and purifying.

5. Virulence test of recombinant fusion proteins in mice

By measuring rFliACCta_NVirulence in mice to verify the actual attenuation of the fusion protein in vivo. And (3) carrying out protein content detection on the purified protein: protein content was determined by BCA Assay (Pierce TM BCA Protein Assay Kit, TG 268883). Should not be less than 0.5 mMg/ml. The protein purity is not lower than 85% by SDS-PAGE detection and gray scanning of the strip. Purified rFliACCta_N16-18 g ICR mice were inoculated via tail vein at different doses, 5 mice were injected per dose, 0.2 mL/mouse, observed for 5 days, and the survival of the mice was recorded.

6. Detection of immune Effect of recombinant fusion proteins

And (3) carrying out protein content detection on the purified protein: protein content was determined by BCA Assay (Pierce TM BCA Protein Assay Kit, TG 268883). Introducing a biphasic oil adjuvant (such as 206 adjuvant) into the oil phase tank, autoclaving at a temperature of at least 121 deg.C for 30min, and cooling to room temperature. According to the protein content measurement result, the purified protein qualified in the test is properly diluted and mixed by PBS (pH value 7.20.01 mol/L). Adding the water phase into an emulsifying tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1(V/V), and stirring for 20-30 min after the addition is finished. Sampling after emulsification, inspecting, subpackaging after being qualified, and finally preparing the vaccine with the concentration of 50 mu g/ml. At the same time, PBS with the same dosage was mixed with adjuvant to be used as control vaccine.

The sub-packaged subunit vaccine is tested according to the appendix of the current Chinese veterinary pharmacopoeia (Chinese veterinary medical Committee, Chinese veterinary pharmacopoeia, two good and one five year edition, China agricultural publishing Co., 2016, hereinafter referred to as Chinese veterinary pharmacopoeia):

(1) traits

The appearance should be a milky white emulsion.

The dosage form should be water-in-oil-in-water (W/O/W). A clean suction pipe is taken, a small amount of vaccine is sucked and dropped on the surface of clean cold water, and the vaccine should spread in a cloud state.

Adding 10ml of the stable suction vaccine into a centrifuge tube, centrifuging for 15min at 3000r/min without demulsification, and separating out water at the bottom of the centrifuge tube with the amount of not more than 0.5 ml.

The viscosity is measured in accordance with the appendix of the Chinese veterinary pharmacopoeia (edited by the Committee of the Chinese veterinary dictionary, the animal pharmacopoeia of the people's republic of China, the 2015 edition, the Chinese agricultural Press, 2016 (hereinafter referred to as the Chinese veterinary pharmacopoeia)), and should meet the regulations.

(2) The sterility test is carried out according to the appendix of Chinese veterinary pharmacopoeia, and the growth should be carried out aseptically.

(3) 2 healthy guinea pigs with the weight of 350-450 g are used for safety inspection, 2.0mL of vaccine is injected subcutaneously, and all the guinea pigs should be healthy and alive after 10 days of observation.

(4) Efficacy test

According to the standard of emphysema gangrene inactivated vaccine in the appendix of Chinese veterinary pharmacopoeia (Chinese veterinary pharmacopoeia committee, Chinese people's republic of China veterinary pharmacopoeia, two good quality and five years edition three parts), the method comprises the following steps:

6 healthy guinea pigs with the weight of 350-450 g were selected, 4 of the guinea pigs were used, 1.0mL of the vaccine was injected intramuscularly, and the remaining 2 guinea pigs were used as injection control vaccines. On 21 days after immunization, 4 guinea pigs in the immunization group and 2 guinea pigs in the control group are taken, at least 0.2ml of clostridium tetani virulent bacterial liquid cultured for 24 hours is injected into muscles respectively, and observation is carried out for 10 days. The control guinea pigs should die all within 72h, and the immunized guinea pigs should protect at least 3.

Examples

The following examples are intended to better illustrate the technical solution of the present invention, but are not intended to limit the technical solution of the present invention.

Example 1 construction, expression and characterization of Escherichia coli BL/MA Strain

1. Gene synthesis

The present application was based on the natural gene sequences according to FliA (C) (SEQ ID NO: 1) and Ccta (SEQ ID NO: 3), and after codon optimization, the coding gene containing FliA (C) and the coding gene containing the middle region (amino acids 95 to 261) of Ccta were connected in series. Meanwhile, the 3' end of the tandem gene is added with an amino acid label coding sequence used for purification, and the gene sequence GFLiACCtA is synthesized by a chemical synthesis method_N(SEQ ID NO: 5). The amino acid sequence is detailed in sequence 6.

Sequence 1

Sequence 3

Sequence 5

Sequence 6

2. Construction of recombinant fusion protein expression vectors

Artificially synthesized GTTc-Tcn alpha_ncAs template, the primer pair 1F/1R (SEQ ID NO: 7/SEQ ID NO: 8) was used) PCR amplification was performed.

Wherein the sequence of the upstream primer 1F is as follows:

5’-ggcatatggc aggcaaatct atg-3 '23 (SEQ ID NO: 7), wherein a restriction enzyme Nde I site and a protective base are introduced into the 5' end of the sequence;

the sequence of the downstream primer 1R is as follows:

5’-ggaagctttt agtggtgatg-3 '20 (SEQ ID NO: 8), and a restriction enzyme HindIII site and a protective base are introduced into the 5' end of the DNA.

The PCR system is as follows:

(Mg2+ plus) 10. mu.l, dNTPs 4. mu.l, upstream and downstream primers 1. mu.l each,

polymerase 1. mu.l, DNA template 2. mu.l, supplemented with ddH₂O to 50. mu.l system. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 2min for 33 cycles; finally, ring extension at 72 ℃ for 10 min.

Recovering the amplified target DNA band, digesting with Nde I/Hind III enzyme, and connecting with pET30a vector digested with the same enzyme to obtain insert GFliactcta_NThe positive clone pET30a-GFLiACCta of (9)_N。

3. Expression of rFliACCta_NConstruction of the genetically engineered Strain of (1)

The extracted plasmid is transformed into escherichia coli BL21(DE3) competent cells, a single clone is selected to be put into an LB liquid culture medium containing kanamycin, the culture is carried out overnight under oscillation at 37 ℃, after the identification by PCR and the target DNA fragment is contained, the strain is named as Escherichia coli BL/MA strain, and equal volume of 50 percent of glycerol LB is added, the strain is frozen and stored at-70 ℃, the strain is delivered to Beijing city Shanghai Wenyao national institute of microbiology, China institute of microbiology, institute No.1 institute of China academy of sciences, China general microbiological culture center, on 03 and 08 days in 2019, the China institute of microbiology, North Asian province, No. 3, the preservation numbers are: CGMCC No. 17319.

Example 2 rFliACCta_NExpression and characterization of

1.rFliACctA_NExpression of

Escherichia coli (E.coli) BL/MA strain was inoculated into 4mL of LB liquid medium containing kanamycin, placed at 37 ℃ and OD₆₀₀When the concentration is 0.6-0.8, IPTG solution with the final concentration of 0.5mM is added and the mixture is respectively placed at 37 ℃ and 15 ℃ for induced culture for 4h and 16 h. After the bacterial liquid culture is finished, the thalli are centrifugally collected, and 10ml of lysis solution (0.02 mol/LTris buffer solution (pH value 7.2) and 0.3mol/LNaCl are added according to the body temperature of each gram of thalli]Resuspending the thallus according to the proportion, and carrying out ultrasonic disruption on the thallus in an ice water bath for 30min under the conditions: the operation time is 9s, the pause time is 9s, and the ultrasonic power is 400W. The crushed bacterial liquid is centrifuged at 12000r/min for 10min at 4 ℃, and the supernatant is collected. mu.L of the supernatant was added to 10. mu.L of 4 XSDS-PAGE loading buffer, and subjected to 12% SDS-PAGE electrophoresis at 70 ℃ for 10min, as shown in FIG. 1. As can be seen from FIG. 1, under the condition of 15 ℃, rFliACCTA_NThe protein exists in the supernatant of thallus lysate in large amount, is expressed in soluble state and accounts for about 30% of the total target protein expression. rFliACCta_NThe optimal induction expression condition of (3) is 15 ℃, and the induction expression is 16 h.

2.rFliACctA_NIdentification of

Adopting the rFliACCtA under the induction condition in the step_NWestern blot identification was performed using anti-His antibodies, and the results are shown in FIG. 2. As can be seen from FIG. 2, rFliACCTA was found in the supernatant of 16h induced cell lysis at 15 ℃_NThe expression level is highest. The spatial structure is closest to the wild-type protein due to the soluble expression of the recombinant fusion protein in the cell lysis supernatant. And further determining the optimal induced expression condition of the target protein to be 15 ℃ by integrating the identification results of SDS-PAGE and Western blot for induced expression for 16 h.

Example 3 rFliACCta_NPurification of (2)

Escherichia coli BL/MA strain was inoculated into 1L LB liquid medium containing kanamycin for fermentation culture, followed by shaking culture at 37 ℃ for OD₆₀₀When the concentration is 0.6-0.8 ℃, the temperature is reduced to 15 ℃, and IPTG solution with the final concentration of 0.5mM is added for induction culture for 16 h. After the bacterial liquid culture is finished, the bacteria are collected by centrifugation for 5min at 5000r/min, and 10ml of lysate (pH value of 7.20.02 mo) is added according to the wet weight of each gram of bacterial/LTris buffer, 0.3mol/LNaCl), and disrupting the cells 3 times at 4 ℃ with a low-temperature high-pressure homogenizer at 800 bar. The lysate is centrifuged at 10000r/min at 4 ℃ for 30min, and the supernatant is collected. rFliACCta which is expressed in a soluble way in the thalli lysis supernatant according to the instruction of the Ni-IDA affinity chromatography medium kit_NAnd (5) purifying. As shown in FIG. 3, the eluate of the 6-7 lanes with higher purity is collected and filtered through a 0.22 μm pore size filter membrane, thus obtaining the primarily purified target protein rFliACCTA_N。

Example 4 rFliACCta_NToxicity test on mice

By measuring rFliACCta_NVirulence in mice to verify whether the recombinant fusion protein is toxic in vivo. Purified rFliACCta_NThree doses of 10. mu.g, 50. mu.g and 100. mu.g, respectively, were inoculated via tail vein into 16-18 g ICR mice, 5 mice per dose were injected, 0.2 ml/mouse. Results mice vaccinated with the above three doses were all healthy and without adverse reactions. The results show that rFliACCTA_NIs non-toxic in mice and is identified as a non-toxic recombinant fusion protein.

Example 5 detection of the immune Effect of recombinant fusion proteins

And (3) carrying out protein content detection on the purified protein: protein content was determined by BCA assay (Pierce TM BCAProteinaassay Kit, TG268883) to obtain the purified protein rFliACCtA_NThe concentration of (A) can reach 0.76 mg/ml. Introducing a biphasic oil adjuvant (such as 206 adjuvant) into the oil phase tank, autoclaving at a temperature of at least 121 deg.C for 30min, and cooling to room temperature. According to the protein content measurement result, the purified protein qualified in the test is properly diluted and mixed by PBS (pH value 7.20.01 mol/L). Adding the water phase into an emulsifying tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1(V/V), and stirring for 20-30 min after the addition is finished. Sampling after emulsification, inspecting, subpackaging after being qualified, and finally preparing the vaccine with the concentration of 50 mu g/ml. At the same time, PBS with the same dosage was mixed with adjuvant to be used as control vaccine.

(1) traits

The appearance was a milky white emulsion.

The dosage form is water-in-oil-in-water (W/O/W). A clean suction pipe is taken, a small amount of vaccine is absorbed and dropped on the surface of clean cold water, and the vaccine is dispersed in a cloud state.

Adding 10ml of the stable suction vaccine into a centrifuge tube, centrifuging for 15min at 3000r/min without demulsification, and separating out water at the bottom of the centrifuge tube with the volume of not more than 0.3 ml.

The viscosity is determined according to the appendix of Chinese animal pharmacopoeia and conforms to the regulations.

(2) The sterility test was performed according to appendix of Chinese veterinary pharmacopoeia, and no bacteria grew.

(3) 2 healthy guinea pigs with the weight of 350-450 g are used for safety inspection, 2.0ml of vaccine is injected subcutaneously for each guinea pig, and the guinea pigs are observed for 10 days to be 2/2 healthy and alive.

(4) Efficacy test

The method is carried out according to the standard of emphysema gangrene inactivated vaccine in the appendix of Chinese veterinary pharmacopoeia, and comprises the following steps:

6 healthy guinea pigs with the weight of 350-450 g were selected, 4 healthy guinea pigs were used, 1.0ml of vaccine was injected intramuscularly, and the remaining 2 healthy guinea pigs were used as injection control vaccines. On 21 days after immunization, 4 guinea pigs of the immunization group and 2 guinea pigs of the control group were taken, and 0.3ml of clostridium tetani virulent bacterial liquid cultured for 24 hours was intramuscular injected respectively, and observed for 10 days. The control guinea pigs died 2/2 within 48h, and the immunized guinea pigs remained 4/4 alive after 10 d.

Thus, the present invention produces rFliACCTA_NThe immunized guinea pigs can resist the attack of the lethal clostridium gangreniformis virulent bacterial liquid under the condition that the antigen content is as low as 50 mu g/mouse.

Sequence listing

<110> China institute for veterinary drug inspection

<120> a strain for avirulent clostridium perfoliatum genetic engineering subunit vaccine and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 429

<212> DNA

<213> Clostridium tetani flagellin (Clostridium chauvoei flagellum)

<400> 1

gcaggtaagt caatggaaaa attaagctca ggtttaagaa taaacagagc tggagatgat 60

gctgcaggac tagcaatctc agaaaaaatg agaggtcaaa ttagaggatt agatcaagca 120

tcaagaaatg ctcaagatgg tatttcatta attcaaacag ctgaaggagc tttaagtgaa 180

actcactcaa ttcttcaaag aatgagagaa ttatcagtac aatcagctaa cgatacaaac 240

gtagctgtag atagaactgc tatccaagat gaaataaact cattaacaga agaaataaac 300

agaatatcag gagatactga attcaatact caaaagttat tagatggtgg tttcaaagga 360

gaattccaaa ttggagctaa ctcaaaccaa acagttaaat tagatatcgg aaacatgagt 420

gctgcaagt 429

<210> 2

<211> 143

<212> PRT

<213> Clostridium tetani flagellin (Clostridium chauvoei flagellum)

<400> 2

Ala Gly Lys Ser Met Glu Lys Leu Ser Ser Gly Leu Arg Ile Asn Arg

1 5 10 15

Ala Gly Asp Asp Ala Ala Gly Leu Ala Ile Ser Glu Lys Met Arg Gly

20 25 30

Gln Ile Arg Gly Leu Asp Gln Ala Ser Arg Asn Ala Gln Asp Gly Ile

35 40 45

Ser Leu Ile Gln Thr Ala Glu Gly Ala Leu Ser Glu Thr His Ser Ile

50 55 60

Leu Gln Arg Met Arg Glu Leu Ser Val Gln Ser Ala Asn Asp Thr Asn

65 70 75 80

Val Ala Val Asp Arg Thr Ala Ile Gln Asp Glu Ile Asn Ser Leu Thr

85 90 95

Glu Glu Ile Asn Arg Ile Ser Gly Asp Thr Glu Phe Asn Thr Gln Lys

100 105 110

Leu Leu Asp Gly Gly Phe Lys Gly Glu Phe Gln Ile Gly Ala Asn Ser

115 120 125

Asn Gln Thr Val Lys Leu Asp Ile Gly Asn Met Ser Ala Ala Ser

130 135 140

<210> 3

<211> 954

<212> DNA

<213> Clostridium tetani cytotoxin A (Clostridium chauvoei flagelum)

<400> 3

atgataaaaa gaatattaat gcttgcttta gcaacaacaa ctatatttag cttaacttta 60

cctttttcct ataaagctgt acaagctcaa gaaaatacat gtatagttga aacaccaagt 120

gaaggagtaa agacttttac atcttcagat actgcttatg cagattataa ttgctttaag 180

actaacttat cagttacatt tattgaagat caacacaata atcaacttac agcacttgta 240

tcaacagaag gatcatttat tccatcagga ttatcacgtg ttggtgggta ttatcaagct 300

gatatgtatt ggccatcaaa atattacaca acattaacaa cttatgatag aaataataga 360

gtaaaaataa ctaaaagtat cccaactaat caaatagata cagtatcagt atcggaaact 420

atgggttata gcattggtgg aagcttatca attgaatatg gaaaagaagg ccctaaagca 480

gggggaggaa taaatggatc atacactgct caaagaagtg taacatatga tcaaccagat 540

tatagaacat tattaatgaa agatagtgta aatagtgcat cttgggaagt tgcttttaat 600

gcaactaaag atggatatga tagagattct tatcatggta tctatggaaa tcaattattt 660

atgagatata gattatataa tacaggaata aataatttaa ctacagataa caatttatct 720

tctttaatag ttggtggttt ttctcctaaa gtagtaattg ctcttacagc accaaaagga 780

actgaagaat caacagttaa agttgaatat aatcgtttta atgatcaata tagattaaga 840

tggtcaggaa ctgaatggta tggagaaaat aatagaaatt ctagaataga tagttcaagt 900

gagtctttca tacttaattg gaaaaaccat actgttgagc atgcaggata ttaa 954

<210> 4

<211> 317

<212> PRT

<213> Clostridium pneumonectasis Cytotoxin A (Clostridium chauvoei Cytotoxin CctA)

<400> 4

Met Ile Lys Arg Ile Leu Met Leu Ala Leu Ala Thr Thr Thr Ile Phe

1 5 10 15

Ser Leu Thr Leu Pro Phe Ser Tyr Lys Ala Val Gln Ala Gln Glu Asn

20 25 30

Thr Cys Ile Val Glu Thr Pro Ser Glu Gly Val Lys Thr Phe Thr Ser

35 40 45

Ser Asp Thr Ala Tyr Ala Asp Tyr Asn Cys Phe Lys Thr Asn Leu Ser

50 55 60

Val Thr Phe Ile Glu Asp Gln His Asn Asn Gln Leu Thr Ala Leu Val

65 70 75 80

Ser Thr Glu Gly Ser Phe Ile Pro Ser Gly Leu Ser Arg Val Gly Gly

85 90 95

Tyr Tyr Gln Ala Asp Met Tyr Trp Pro Ser Lys Tyr Tyr Thr Thr Leu

100 105 110

Thr Thr Tyr Asp Arg Asn Asn Arg Val Lys Ile Thr Lys Ser Ile Pro

115 120 125

Thr Asn Gln Ile Asp Thr Val Ser Val Ser Glu Thr Met Gly Tyr Ser

130 135 140

Ile Gly Gly Ser Leu Ser Ile Glu Tyr Gly Lys Glu Gly Pro Lys Ala

145 150 155 160

Gly Gly Gly Ile Asn Gly Ser Tyr Thr Ala Gln Arg Ser Val Thr Tyr

165 170 175

Asp Gln Pro Asp Tyr Arg Thr Leu Leu Met Lys Asp Ser Val Asn Ser

180 185 190

Ala Ser Trp Glu Val Ala Phe Asn Ala Thr Lys Asp Gly Tyr Asp Arg

195 200 205

Asp Ser Tyr His Gly Ile Tyr Gly Asn Gln Leu Phe Met Arg Tyr Arg

210 215 220

Leu Tyr Asn Thr Gly Ile Asn Asn Leu Thr Thr Asp Asn Asn Leu Ser

225 230 235 240

Ser Leu Ile Val Gly Gly Phe Ser Pro Lys Val Val Ile Ala Leu Thr

245 250 255

Ala Pro Lys Gly Thr Glu Glu Ser Thr Val Lys Val Glu Tyr Asn Arg

260 265 270

Phe Asn Asp Gln Tyr Arg Leu Arg Trp Ser Gly Thr Glu Trp Tyr Gly

275 280 285

Glu Asn Asn Arg Asn Ser Arg Ile Asp Ser Ser Ser Glu Ser Phe Ile

290 295 300

Leu Asn Trp Lys Asn His Thr Val Glu His Ala Gly Tyr

305 310 315

<210>5

<211>1704

<212>DNA

<213> Clostridium tetani flagellin and cytotoxin A fusion protein (Clostridium chauvoei GFliACCtan)

<400>5

catatggcag gcaaatctat ggagaaactg agcagcggtc tgcgtatcaa ccgtgctggc 60

gacgatgccg caggtctggc gatcagcgag aagatgcgtg gtcagatccg tggcctggac 120

caggcaagcc gtaacgcgca agacggtatt tccctgatcc agaccgcaga aggtgcactg 180

tccgagactc atagcattct gcaacgtatg cgtgagctgt ccgtgcagtc tgccaacgat 240

actaacgttg cggttgaccg taccgccatc caggacgaaa tcaacagcct gaccgaggag 300

atcaatcgta tctccggtga cactgagttc aacactcaga aactgctgga cggtggcttc 360

aaaggcgagt tccagattgg cgctaacagc aaccagaccg ttaaactgga tattggcaat 420

atgtccgctg catctctggg tctgaccacc actaactccc tggagagcaa ggctctggcg 480

ctgaacagca atctggcaga cggtagctac aagattagcg gcactaacct ggtggatact 540

aatggtaaca ctgttggcac cttcaactct ggtgctaaga agatcgtagt gaacggtcag 600

gacactgtgt tcactaaagc tgcactggca gatggtgcag ttctgaccgt gaaaggcggt 660

attgccgaca tcaagaacac tatgactggt gcggctaaga agctgtccag cggctcttat 720

gaaatctccg gtactaacgt tatcaaagat ggcaaactgg ttggcacttt cgcaactggc 780

gctaagaaac tgaccatcga cggcgtgggc gatgtaaccg aggctgaact gggcttcatt 840

actgatcaga tgaaagacgg cgtgaaattc actatcaacg gtagcgacgt gtccacccgt 900

gagctggcat ctggctctat caagaccatc aactccgcta ttgaacaagt aagcactcag 960

cgttccaaac tgggtgcagt tcagaaccgc ctggaacaca ctatcaacaa tctgaacact 1020

tctagcgaga acctgaccgc tgccgagtct cgtgttcgtg atgttgacat ggcgaaagaa 1080

atgatggcgt tctctaagaa taacatcctg tcccaagcag cgcaggcgat gctgggtcag 1140

gcaaaccagc agccgcaggg cggtggtggc ggttccggtg gctattacca ggctgatatg 1200

tactggccga gcaaatacta taccactctg accacctatg atcgtaacaa ccgcgtaaag 1260

atcaccaaat ccattccgac caaccaaatt gataccgtgt ccgtatctga aactatgggt 1320

tacagcatcg gtggttctct gagcatcgaa tacggcaagg aaggtccgaa agcaggtggt 1380

ggcatcgacg gctcctacac cgctcaacgt tctgttacct atgatcagcc ggactatcgt 1440

actctgctga tgaaagattc cgttaacagc gcttcctggg aggttgcctt taacgctacc 1500

aaagatggtt acgatcgtga cagctaccac ggcatctacg gcaaccagct gtttatgcgc 1560

tatcgtctgt acaacaccgg catcaacaac ctgaccaccg acaacaatct gagcagcctg 1620

atcgtaggcg gtttctctcc gaaagtggtt atcgccctga ccgcaccgaa aggcactcac 1680

caccaccacc atcactaaaa gctt 1704

<210>6

<211>565

<212>PRT

<400>6

His Met Ala Gly Lys Ser Met Glu Lys Leu Ser Ser Gly Leu Arg Ile

1 5 10 15

Asn Arg Ala Gly Asp Asp Ala Ala Gly Leu Ala Ile Ser Glu Lys Met

20 25 30

Arg Gly Gln Ile Arg Gly Leu Asp Gln Ala Ser Arg Asn Ala Gln Asp

35 40 45

Gly Ile Ser Leu Ile Gln Thr Ala Glu Gly Ala Leu Ser Glu Thr His

50 55 60

Ser Ile Leu Gln Arg Met Arg Glu Leu Ser Val Gln Ser Ala Asn Asp

65 70 75 80

Thr Asn Val Ala Val Asp Arg Thr Ala Ile Gln Asp Glu Ile Asn Ser

85 90 95

Leu Thr Glu Glu Ile Asn Arg Ile Ser Gly Asp Thr Glu Phe Asn Thr

100 105 110

Gln Lys Leu Leu Asp Gly Gly Phe Lys Gly Glu Phe Gln Ile Gly Ala

115 120 125

Asn Ser Asn Gln Thr Val Lys Leu Asp Ile Gly Asn Met Ser Ala Ala

130 135 140

Ser Leu Gly Leu Thr Thr Thr Asn Ser Leu Glu Ser Lys Ala Leu Ala

145 150 155 160

Leu Asn Ser Asn Leu Ala Asp Gly Ser Tyr Lys Ile Ser Gly Thr Asn

165 170 175

Leu Val Asp Thr Asn Gly Asn Thr Val Gly Thr Phe Asn Ser Gly Ala

180 185 190

Lys Lys Ile Val Val Asn Gly Gln Asp Thr Val Phe Thr Lys Ala Ala

195 200 205

Leu Ala Asp Gly Ala Val Leu Thr Val Lys Gly Gly Ile Ala Asp Ile

210 215 220

Lys Asn Thr Met Thr Gly Ala Ala Lys Lys Leu Ser Ser Gly Ser Tyr

225 230 235 240

Glu Ile Ser Gly Thr Asn Val Ile Lys Asp Gly Lys Leu Val Gly Thr

245 250 255

Phe Ala Thr Gly Ala Lys Lys Leu Thr Ile Asp Gly Val Gly Asp Val

260 265 270

Thr Glu Ala Glu Leu Gly Phe Ile Thr Asp Gln Met Lys Asp Gly Val

275 280 285

Lys Phe Thr Ile Asn Gly Ser Asp Val Ser Thr Arg Glu Leu Ala Ser

290 295 300

Gly Ser Ile Lys Thr Ile Asn Ser Ala Ile Glu Gln Val Ser Thr Gln

305 310 315 320

Arg Ser Lys Leu Gly Ala Val Gln Asn Arg Leu Glu His Thr Ile Asn

325 330 335

Asn Leu Asn Thr Ser Ser Glu Asn Leu Thr Ala Ala Glu Ser Arg Val

340 345 350

Arg Asp Val Asp Met Ala Lys Glu Met Met Ala Phe Ser Lys Asn Asn

355 360 365

Ile Leu Ser Gln Ala Ala Gln Ala Met Leu Gly Gln Ala Asn Gln Gln

370 375 380

Pro Gln Gly Gly Gly Gly Gly Ser Gly Gly Tyr Tyr Gln Ala Asp Met

385 390 395 400

Tyr Trp Pro Ser Lys Tyr Tyr Thr Thr Leu Thr Thr Tyr Asp Arg Asn

405 410 415

Asn Arg Val Lys Ile Thr Lys Ser Ile Pro Thr Asn Gln Ile Asp Thr

420 425 430

Val Ser Val Ser Glu Thr Met Gly Tyr Ser Ile Gly Gly Ser Leu Ser

435 440 445

Ile Glu Tyr Gly Lys Glu Gly Pro Lys Ala Gly Gly Gly Ile Asp Gly

450 455 460

Ser Tyr Thr Ala Gln Arg Ser Val Thr Tyr Asp Gln Pro Asp Tyr Arg

465 470 475 480

Thr Leu Leu Met Lys Asp Ser Val Asn Ser Ala Ser Trp Glu Val Ala

485 490 495

Phe Asn Ala Thr Lys Asp Gly Tyr Asp Arg Asp Ser Tyr His Gly Ile

500 505 510

Tyr Gly Asn Gln Leu Phe Met Arg Tyr Arg Leu Tyr Asn Thr Gly Ile

515 520 525

Asn Asn Leu Thr Thr Asp Asn Asn Leu Ser Ser Leu Ile Val Gly Gly

530 535 540

Phe Ser Pro Lys Val Val Ile Ala Leu Thr Ala Pro Lys Gly Thr His

545 550 555 560

His His His His His

565

<210> 7

<211> 23

<212> DNA

<213> Artificial Synthesis of primer (1F)

<400> 7

ggcatatggc aggcaaatct atg 23

<210> 8

<211> 20

<212> DNA

<213> Artificial Synthesis of primer 1R (1R)

<400> 8

ggaagctttt agtggtgatg 20

Claims

1. A non-toxic bacillus pyogenes gene engineering subunit vaccine strain is characterized in that the strain is a recombinant fusion protein (rFliACCTA) for recombinant expression of clostridium pyogenes flagellin (C) and cytotoxin A (Ccta)_N) Escherichia coli (BL 21) (DE3) cell of (E.coli), which was designated as Escherichia coli BL/MA strain;

the recombinant fusion protein rFliACCtA expressed by the strain_NContains flagellin FliA (C) and cytotoxin A (CctA); compared with wild type cytotoxin A (CctA), only contains nontoxic amino acid segments from 95 th to 261 th;

the recombinant fusion protein rFliACCta_NNon-toxicity; the fusion protein rFliACCta expressed by the strain_NThe C-terminus of which contains a tag 6 histidine (6 × His) tag to facilitate protein purification;

the strain is delivered to the general microorganism center of China Committee for culture Collection, China academy of sciences, microorganism research institute No. 3, Xilu No.1, Beijing, Chaoyang, 03.08 days in 2019, and the preservation number is: CGMCC No. 17319.

2.The use of the strain of claim 1 for the preparation of a non-toxic clostridium emphysema genetic engineering subunit vaccine, wherein said vaccine is prepared by the steps of: the expression of the fusion protein rFliACCta is used_NThe Escherichia coli BL/MA strain is used as a vaccine production strain, and is prepared by fermentation culture, induction expression, thallus crushing, soluble antigen protein separation and purification, and then adding an adjuvant and mixing.