A non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine and its production method

Abstract

The present invention relates to a non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine and a production method thereof. The fusion protein vaccine prepared by the present invention is produced by using a fusion protein of a Clostridium perfringens ε toxin containing 3 amino acid mutations after codon optimization and a Clostridium putrefaciens α toxin containing 4 amino acid mutations and 11 amino acid deletions, that is, the integrity and spatial conformation of the natural toxin are retained to the maximum extent, thereby maintaining its immunogenicity, and avoiding the biosafety risks caused by a single amino acid mutation. The vaccine also has the advantages of simple preparation process and excellent vaccine efficacy. Compared with the currently commercialized sheep rapid disease and sheep enterotoxemia inactivated vaccines in my country, the biosafety risk in the vaccine production process is greatly reduced. It is an ideal candidate vaccine for upgrading the current Clostridium perfringens and Clostridium putrefaciens disease vaccines in my country; and when preparing a combined vaccine with other antigens, the combined vaccine can be prepared without increasing the dosage of the combined vaccine.

Description

Technical Field

[0001] The invention relates to a non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine and a production method thereof, belonging to the field of veterinary biological products. Background Art

[0002] Clostridium perfringens and Clostridium putrefaciens are anaerobic bacteria that can cause disease in humans and a variety of animals, and are extremely harmful to human health and livestock and poultry farming. Among them, Clostridium perfringens is one of the main pathogens of traumatic gas gangrene and human food poisoning, as well as sheep anthrax, lamb dysentery, cattle and sheep necrotic enteritis, and cattle and sheep enterotoxemia, while Clostridium putrefaciens is the main pathogen of sheep rapid disease. The pathogenic factors of both Clostridia are exotoxins secreted by the bacteria. Clostridium perfringens can be divided into five toxin types, namely A, B, C, D and E, according to the types of the four main lethal exotoxins α (CPA), β (CPB), ε (ETX) and ι (CPI) produced (Revitt-Mills, S; Rood, J; Adams, V. Clostridium perfringens extracellular toxins and enzymes: 20 and counting. Microbiol. Aust. 2015, 36, 114-117.), while Clostridium septicum has only one key lethal virulence factor, namely α toxin (Tweten R K. Clostridium perfringens beta toxin and Clostridium septicum alpha toxin: their mechanisms and possible role in pathogenesis [J]. Vet Microbiol, 2001, 82 (1): 1-9.). Since Clostridium perfringens and Clostridium putrefaciens diseases have the characteristics of rapid onset, short course and extremely high mortality, once the disease occurs, sudden death often occurs due to exotoxin poisoning before treatment is available, so vaccination is an effective method for preventing and controlling the disease (Lu Chengping. Veterinary Microbiology [M]. Beijing: China Agriculture Press, 2013: 194-196.). At present, the commercial vaccines used for the above two diseases are mainly inactivated vaccines. Although such vaccines have achieved certain effects in preventing diseases caused by Clostridium perfringens and Clostridium putrefaciens in animals, these vaccines still expose some defects during use, such as vaccine immunization easily causing local inflammation and toxic reactions in animals; the inactivation of exotoxins involved in the preparation process has biosafety risks such as toxin leakage or incomplete inactivation; in addition, various microtoxins in the culture supernatant and bacterial metabolites often become allergens for the immunized animals, and the vaccinated animals are prone to adverse reactions, resulting in a decrease in the immune effect or even immune failure. Therefore, developing genetically engineered vaccines against Clostridium perfringens and Clostridium putrefaciens toxins with good safety, high effective antigen content and strong immunogenicity is the future development direction.

[0003] At present, the commercial inactivated clostridial vaccines with large market usage are mainly triple-four-protection inactivated vaccines for sheep blight, anthrax, lamb dysentery, and enterotoxemia, as well as multi-component dry powder vaccines for sheep clostridial disease. Among them, sheep blight is mainly caused by alpha toxin (CSA) secreted by Clostridium perfringens (Tweten R K. Clostridium perfringens beta toxin and Clostridium septicum alpha toxin: their mechanisms and possible role in pathogenesis [J]. Vet Microbiol, 2001, 82 (1): 1-9.), and enterotoxemia is mainly related to epsilon toxin (ETX) secreted by type D Clostridium perfringens.

[0004] CSA is composed of 440 or 443 amino acids, which are mainly divided into three domains: D1, D2 and D3. During its maturation process, 29 or 32 amino acid sites of the N segment will be removed, leaving only 411 amino acids. Among them, the D1 domain is a complex multifunctional domain that plays a role in receptor binding, oligomerization and pore formation. Among the mutants with mutations in 45 key amino acid sites in this domain, 17 mutants lost their cell hemolytic activity, while their cell binding activities were different. The D2 domain is mainly involved in the pore formation process, while the D3 domain is mainly involved in the oligomerization of toxin molecules. Existing studies have found that deleting 11 amino acids (positions 212 to 222) in the amphipathic β-hairpin transmembrane region of the D2 region does not significantly change the normal folding of CSA, but completely loses its cytotoxicity and its role in Clostridium septicum disease, while the antigenicity of the recombinant toxin has not been verified (Kennedy CL, Lyras D, Cordner LM, et al. Pore-forming activity of alpha-toxin is essential for clostridium septicum-mediated myonecrosis [J]. Infection & Immunity, 2009, 77 (3): 943-951.).

[0005] ETX is mainly produced by B-type and D-type Clostridium perfringens, with a total length of 296 amino acids, and is secreted outside the bacterium in the form of protoxin. After the protoxin is acted on by the host's trypsin and chymotrypsin or the protease of Clostridium itself, 11-13 amino acids at the N-terminus and 22-29 amino acids at the C-terminus are removed, thereby activating into mature toxin. The toxin belongs to the β-perforin family member and is the most toxic toxin among Clostridium perfringens toxins, and its toxicity is second only to botulinum toxin and tetanus neurotoxin (Li JH, Adams V, Bannam TL, Miyamoto K, Garcia JP, Uzal FA, Rood JL, McClanea BA. Toxinplasmids ofclostridium perfringens, Microbiology and Molecular Biology Reviews, 2013, 77 (2): 208-233.). Therefore, achieving attenuation or even avirulence of toxins and constructing related attenuated or avirulent bodies are particularly important for the development of toxin genetic engineering subunit vaccines and the research and application of multivalent subunit vaccines with toxins as antigenic components. Although there are reports of attenuated mutants of Clostridium perfringens ε toxin, such as Yu Li et al. of Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences ("Attenuated mutants of Clostridium perfringens ε toxin and its application", application number: 201410707351.6) mutated the 71st tyrosine of the mature toxin of wild-type Clostridium perfringens ε toxin to a non-aromatic amino acid, and obtained an ε toxin mutant attenuated for cells or animals. However, since the application only mutated one amino acid, it is easy to reverse mutation and become a highly virulent toxin again, which brings serious biosafety risks to the actual large-scale production of vaccines in the future. Song Xiaohui et al. from the China Animal Disease Prevention and Control Center ("Recombinant ε protein for inhibiting Clostridium perfringens infection, its preparation method and application", application number: 201610301989.9) designed three recombinant ε toxin proteins, but all of them lacked the first 50 amino acid residues of the wild-type ε toxin protein. This strategy of using protein fragments as immunogens is likely to result in incomplete antigenicity of the antigen protein, thereby affecting its immunogenicity. Summary of the invention

[0006] The present invention aims to prepare a fusion protein (rETX) of a mutant of Clostridium perfringens and a mutant of Clostridium putrefaciens CSA. m3 CSA M4△11 ), on the one hand, it maximizes the immunogenicity of the corresponding toxin, and avoids the biosafety risks caused by single amino acid mutations. At the same time, the vaccine also has the advantages of simple preparation process, low immune dose, and good immune efficacy, and is used to prevent sheep rapid disease and enterotoxemia.

[0007] Technical solution of the present invention

[0008] 1. A non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine, characterized in that the fusion protein contained in the vaccine is produced by recombinantly expressing rETX m3 CSA M4△11 The strain was prepared from Escherichia coli BL21 (DE3), named as Escherichia coli BL / EC strain, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on January 11, 2019, with the deposit number: CGMCC No.17165;

[0009] The coding gene of the fusion protein is constructed by concatenating the ETX coding gene containing multiple amino acid mutations and the CSA coding gene containing multiple amino acid mutations and amino acid deletions based on the natural gene sequences of Clostridium perfringens ETX and Clostridium putrefaciens CSA after codon optimization, and adding an amino acid tag coding gene at the C-terminus to obtain a fusion protein that is non-toxic to animals through host cell expression.

[0010] 2. The Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine of the present invention is characterized in that the fusion protein rETX m3 CSA M4△11 Compared with the mature ETX of wild-type Clostridium perfringens, it contains mutations of amino acid residues at both the N-terminus and the C-terminus and multiple amino acids, preferably 3 amino acid mutations, namely, tyrosine at position 30 mutates to alanine Y30A, histidine at position 106 mutates to proline H106P, and tyrosine at position 196 mutates to alanine Y196A; compared with the mature CSA of wild-type Clostridium putrefaciens, the D1 domain contains multiple amino acid mutations, preferably 4 amino acid mutations, namely, cysteine ​​at position 86 mutates to leucine C86L, asparagine at position 296 mutates to alanine N296A, histidine at position 269 mutates to alanine H301A, and tryptophan at position 310 mutates to alanine W342A. Meanwhile, the D2 domain contains multiple amino acid deletions, preferably 11 amino acid deletions, namely, amino acids at positions 212 to 222.

[0011] 3. The Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine of the present invention is characterized in that the fusion protein rETX m3 CSA M4△11 Its coding gene sequence has been codon-optimized, making it easier to achieve high expression in Escherichia coli.

[0012] 4. The Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine of the present invention is characterized in that the fusion protein rETXm3 CSA M4△11 It is a non-toxic protein, which greatly reduces the biosafety risk in the vaccine production process.

[0013] 5. The Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine of the present invention is characterized in that the fusion protein is expressed in a soluble form in Escherichia coli BL (DE3), which can retain the spatial conformation of the natural toxin protein to the maximum extent, thereby maintaining its immunogenicity; it also avoids the influence of the cumbersome process of inclusion body denaturation and renaturation on the immunogenicity of the antigen protein, thereby reducing the preparation time and production cost of the vaccine.

[0014] 6. The Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine of the present invention is characterized in that the fusion protein has an amino acid tag that is convenient for purification, and the optimized one is in rETX m3 CSA M4△11 The C-terminus of the protein contains a 6-histidine (6*His) tag to facilitate protein purification.

[0015] 7. The fusion protein vaccine of Clostridium perfringens and Clostridium putrefaciens of the present invention is characterized in that its preparation method is: the fusion protein is expressed by a host cell. As used herein, the term "host cell" covers prokaryotic cells and / or eukaryotic cells, and can be Clostridium perfringens, Clostridium acetobutylicum, Bacillus cereus, Bacillus thuringiensis, Bacillus seuformis, Bacillus thermophilus, Bacillus anthracis, Bacillus megaterium, Bacillus subtilis, Escherichia coli or yeast cells. Preferably, the host cell is an Escherichia coli host cell, in particular Escherichia coli BL21 (DE3) or Rosetta (DE3). The present invention uses a host cell expressing the fusion protein rETX. m3 CSA M4△11 The Escherichia coli BL21 (DE3), named BL / EC strain, is used as the vaccine production strain. After fermentation culture, induced expression, bacterial disruption, and soluble antigen protein separation and purification, it is mixed with a biphasic oil-emulsion adjuvant.

[0016] Beneficial effects of the present invention

[0017] The present invention relates to a non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine and a production method thereof. The present invention comprises an ETX mutant (containing both the N-terminal and C-terminal amino acid residues and three amino acid mutations, referred to as ETX for short) m3 ) and CSA mutant (containing 4 amino acid mutations and 11 amino acid deletions, referred to as CSA M4△11 ) were expressed in tandem to obtain the fusion protein rETX that is non-toxic to mice m3 CSA M4△11The fusion protein is expressed soluble in E. coli BL (DE3), which can not only retain the spatial conformation of the natural toxin to the maximum extent, thereby maintaining its immunogenicity, but also avoid the biosafety risks caused by single amino acid mutations, and show good safety and immune protection in the rabbit model. Compared with the currently commercialized inactivated Clostridium difficile vaccine in my country, this vaccine has the advantages of low immunization dose, good immune efficacy, simple vaccine production process, and greatly reduced biosafety risks in the vaccine production process. In addition, the serum neutralization titer of the prepared vaccine after one immunization is much higher than the current commercial vaccine standard, and it is an ideal candidate vaccine for upgrading the current Clostridium difficile vaccine in my country.

[0018] In view of the fact that the existing commercial clostridial vaccines in my country must be inactivated and detoxified with formaldehyde, there are potential biosafety risks, which also affect the safety of the vaccine in the field; at the same time, the current commercial vaccines also have the problem of unstable toxin production during the production process, resulting in unstable vaccine efficacy. Therefore, the vaccine produced in this application is an ideal candidate vaccine for upgrading the current inactivated Clostridium ovis vaccine in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :rETX m3 CSA M4△11 SDS-PAGE identification results of expression, M1: Protein marker; 1: BSA (1μg); 2: BSA (2μg); 3: cell lysate induced by empty vector pET30a; 4: cell lysate induced by target plasmid; 5: cell lysis supernatant induced by empty vector pET30a; 6: cell lysis supernatant induced by target plasmid; 7: cell lysis precipitate induced by empty vector pET30a; 8: cell lysis precipitate induced by target plasmid.

[0020] Figure 2 :rETX m3 CSA M4△11 Western blot (using anti-His antibody) identification results of expression: M2: Western blot marker; 1: cell lysate induced by empty vector pET30a; 2: cell lysate induced by target plasmid; 3: cell lysis supernatant induced by target plasmid; 4: cell lysis precipitate induced by target plasmid.

[0021] Figure 3 :rETX m3 CSA M4△11Purification M1: Protein marker; M1: Protein marker; 1: Cell lysate after IPTG induction; 2: Cell lysate supernatant after IPTG induction; 3: Flow-through; 4: Elution solution of 20mM Imidazole; 5: Elution solution of 50mM Imidazole; 6: Elution solution of 300mM Imidazole; 7: Ni-IDA medium.

[0022] Figure 4 :rETX m3 CSA M4△11 Neutralization titers of immune rabbit and sheep sera against toxins (1): type D toxin of Clostridium perfringens; 2: toxin of Clostridium putrefaciens.

[0023] The present invention relates to biological material resource information

[0024] The microorganisms involved in the present invention are: expressing an ETX mutant containing 3 amino acid mutations, a CSA mutant containing 4 amino acid mutations and 11 amino acid deletions, and a fusion protein rETX containing a 6-histidine (6*His) tag at the C-terminus of the fusion protein m3 CSA M4△11 The strain was prepared from Escherichia coli BL21 (DE3), which was named Escherichia coli BL / EC strain (strain). The strain was sent to the General Microbiology Center of China Microbiological Culture Collection Administration Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on January 11, 2019, and the deposit number is: CGMCC No.17165.

[0025] Specific implementation methods of the present invention

[0026] 1. Construction of the expression vector for the fusion protein of non-toxic Clostridium perfringens and Clostridium putrefaciens

[0027] (1) Gene synthesis: Based on the natural gene sequences of Clostridium perfringens ETX and Clostridium putrefaciens CSA, after codon optimization, the ETX encoding gene containing multiple amino acid mutations and the CSA encoding gene containing multiple amino acid mutations and amino acid deletions were connected in series. At the same time, the amino acid tag encoding gene used for purification was added to the C-terminus of the fusion protein. The gene sequence (Sequence 1 and Sequence 2) was synthesized by chemical synthesis.

[0028] (1) Construction of fusion protein expression vector

[0029] The synthetic gene was used as a template, and the designed primer pair (sequence 3 and sequence 4) was used to amplify the target DNA band by PCR. After recovery, it was digested with the prokaryotic expression vector by double enzyme digestion and then connected to obtain the prokaryotic expression vector with the target gene inserted. The connected plasmid was transformed into DH5α competent cells, and a single clone was picked and placed in LB liquid culture medium containing kanamycin, and cultured at 37°C overnight with shaking, and the plasmid was extracted for use.

[0030] 2. Construction of genetically engineered strains expressing fusion proteins.

[0031] The extracted plasmid was transformed into competent E. coli BL21 (DE3) cells, and a single clone was picked and placed in LB liquid medium containing kanamycin. It was cultured at 37°C overnight with shaking. After PCR identification, positive strains containing the target DNA fragment were obtained, and an equal volume of 50% glycerol LB was added and frozen at -70°C.

[0032] 3. Preparation of fusion protein vaccine

[0033] (1) Bacterial strain: The bacterial strain used for vaccine production is the BL / EC strain of Escherichia coli expressing the fusion protein.

[0034] (2) Primary seed propagation and identification: The freeze-dried bacteria were thawed with a small amount of LB liquid medium, streaked onto a solid LB plate containing kanamycin, and cultured at 37°C for 12 to 16 hours. Single colonies that met the standards were selected and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C for 8 to 12 hours. The colonies were mixed with 50% glycerol in equal proportions and packaged, and stored at below -15°C. After passing the purity test, they were used as primary seeds for seedling production.

[0035] (3) Secondary seed propagation and identification: Take the primary seeds, inoculate 1% of them into LB liquid culture medium containing kanamycin, and culture them at 37°C with shaking for 8 to 12 hours to obtain the secondary seeds.

[0036] (4) Preparation of antigen for seedling production: Take qualified secondary seeds and inoculate LB liquid medium containing kanamycin at 2% of the total volume of the medium. The culture temperature is 37°C and the pH value is 7.0. When the culture OD 600 When the value was 1.2-1.6, IPTG was added at a final concentration of 0.5 M to induce culture for 4 h.

[0037] (5) Bacterial disruption: Collect the cells by centrifugation at 5000 r / min for 5 min, resuspend the cells in 10 mL of lysis buffer (pH 7.2, 0.02 mol / L Tris buffer, 0.3 mol / L NaCl) per gram of wet weight of the cells, disrupt the cells using a high-pressure homogenizer at a pressure of 800 bar, and collect the supernatant by centrifugation.

[0038] (6) Purification: The soluble target protein in the supernatant of bacterial lysis was purified according to the instruction manual of the Ni-IDA affinity chromatography medium kit (Nanjing GenScript), filtered through a 0.22 μm pore size filter membrane, and stored at -80°C.

[0039] (7) Protein content detection: The protein content was detected by BCA method (PierceTM BCA Protein Assay Kit, TG268883).

[0040] (8) Sterility test: According to the appendix of the current Chinese Pharmacopoeia (Chinese Pharmacopoeia Committee, Pharmacopoeia of the People's Republic of China, 2015 Edition Part III, China Agriculture Press, 2016, hereinafter referred to as the Chinese Pharmacopoeia). Sterile growth should be observed.

[0041] (7) Vaccine preparation: introduce the biphasic oil adjuvant (such as 201 adjuvant) into the oil phase tank, sterilize it under high pressure at at least 121°C for 30 minutes, and cool it to room temperature for use. According to the results of the protein content determination, dilute the qualified purified protein with PBS (pH 7.20.01 mol / L) at a certain ratio. Add the diluted water phase to the emulsification tank respectively, stir at 80-100r / min, and slowly add the oil phase at a ratio of 1:1 (V / V). Stir for 20-30 minutes after adding. Take samples after emulsification for inspection, and package them after passing the test.

[0042] 4. Testing of non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccines

[0043] (1) Properties

[0044] Appearance: milky white emulsion.

[0045] The dosage form is water-in-oil-in-water (W / O / W). Take a clean pipette and draw a small amount of vaccine onto the surface of clean cold water. It should spread in a cloud-like manner.

[0046] Stability: Pipette 10mL of vaccine into a centrifuge tube and centrifuge at 3000r / min for 15min. The emulsion should not break and the water precipitated at the bottom of the tube should not exceed 0.5mL.

[0047] The viscosity shall be measured according to the appendix of the Chinese Pharmacopoeia of Veterinary Medicine and shall comply with the regulations.

[0048] (2) The filling quantity inspection shall be carried out in accordance with the appendix of the Chinese Pharmacopoeia of Veterinary Medicine and shall comply with the regulations.

[0049] (3) Sterility test shall be carried out according to the appendix of the Chinese Pharmacopoeia of Veterinary Medicine, and sterile growth shall be observed.

[0050] (4) Safety test: 4 healthy rabbits weighing 1.5-2.0 kg were injected intramuscularly or subcutaneously with 4.0 mL of the vaccine and observed for 10 days. All rabbits should be alive and well.

[0051] (5) Effectiveness test

[0052] Serum neutralization method: Select 4 healthy rabbits weighing 1.5-2.0 kg and 4 sheep aged 6-12 months and weighing 30-40 kg, whose neutralization titers to D-type Clostridium perfringens toxin and Clostridium putrefaciens toxin are less than 1, that is, they are all antibody-negative, and inject 2.0 mL of the vaccine subcutaneously or intramuscularly into the neck. 14-21 days after vaccination, blood is collected, serum is separated, and 0.4 mL of each is mixed with D-type Clostridium perfringens toxin (containing 12 or more mouse MLD) and Clostridium putrefaciens (containing 4 or more mouse MLD), and then placed at 37°C for 40 minutes, and then injected intravenously into 2 mice of 16-18 g, 0.3 mL / mouse. At the same time, 2 mice from the same batch were injected with 1 MLD of the same toxin as the toxin serum mixture as a control. The mice tested for Clostridium putrefaciens were observed for 3 days, and the mice tested for D-type Clostridium perfringens were observed for 1 day, and the results were determined.

[0053] All the control mice died, and the serum neutralization titers against type D Clostridium perfringens and Clostridium putrefaciens toxins reached 3 or above (0.1 mL of immune animal serum neutralized 3 or more MLD toxins) and 1 or above (0.1 mL of immune animal serum neutralized 1 or more MLD toxins), respectively, and were judged to be qualified.

[0054] Immunization challenge method: 12 healthy rabbits weighing 1.5-2.0 kg were divided into two groups, each with 6 rabbits. 4 rabbits in each group were injected with 2.0 mL of vaccine subcutaneously or intramuscularly in the neck. 14-21 days after vaccination, 1 MLD of D-type Clostridium perfringens and Clostridium putrefaciens toxin were injected intravenously, respectively, and observed for 3-5 days. All rabbits in the control group should die, and at least 3 rabbits in the immunization group should be protected. Example

[0055] The following examples are provided to better illustrate the technical solution of the present invention, but are not intended to limit the technical solution of the present invention.

[0056] Example 1

[0057] ——Construction and identification of BL / EC strain of Escherichia coli

[0058] 1. Gene Synthesis

[0059] According to the natural gene sequences of Clostridium perfringens ETX and Clostridium putrefaciens CSA, after codon optimization, the ETX coding gene containing both N-terminal and C-terminal amino acid residues and 3 amino acid mutations (Y30A, H106P and Y196A) and the CSA coding gene containing 4 amino acid mutations (C86L, N296A, H301A and W342A) and 11 amino acid deletions were connected in series. At the same time, the coding gene of the 6×His tag was added to the C-terminus of the fusion protein. The gene sequence GETX was synthesized by chemical synthesis. m3 CSA M4△11 , contains 2166 nucleotides in total, and the corresponding protein is called rETX m3 CSA M4△11 The specific nucleic acid sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0060] Sequence 1:

[0061]

[0062]

[0063] Sequence 2:

[0064]

[0065]

[0066] 2. Expression of rETX m3 CSA M4△11 Construction of prokaryotic expression vector

[0067] Synthetic GETX m3 CSA M4△11 As a template, PCR amplification was performed using primer pair 1F / 1R (sequence 3 / sequence 4). The sequence of the upstream primer 1F is:

[0068] 5'-gg catatg aa agaaatctcc aac-3'23 (sequence 3), with a restriction endonuclease NdeⅠ site and protective bases introduced at the 5' end;

[0069] The sequence of downstream primer 1R is:

[0070] 5'-gg aagctt tt agtggtgatg at-3'22 (sequence 4), a restriction endonuclease HindIII site and protective bases were introduced at its 5' end.

[0071] The PCR system is: Buffer (Mg2+plus) 10μL, dNTPs 4μL, upstream and downstream primers 1μL each, 1 μL HS polymerase, 2 μL DNA template, ddH2O added to 50 μL system. PCR reaction conditions are: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, 30 s, a total of 33 cycles; finally 72℃ extension for 10 min.

[0072] The amplified target DNA band was recovered, digested with NdeⅠ / HindIII, and connected with the pET30a vector digested with the same enzymes to obtain the inserted GETX m3 CSA M4△11 Gene positive clone pET30a-ETX m3 CSA M4△11 The ligated plasmid was transformed into DH5α competent cells, and a single clone was picked and placed in LB liquid medium containing kanamycin, cultured at 37°C with shaking overnight, and the plasmid was extracted for later use.

[0073] 3. Expression of rETX m3 CSA M4△11 Construction of genetically engineered bacteria BL / EC strain

[0074] The extracted plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and a single clone was picked out and placed in LB liquid medium containing kanamycin. It was cultured at 37°C with shaking overnight. After PCR identification, the strain containing the target DNA fragment was named Escherichia coli BL / EC strain, and an equal volume of 50% glycerol LB was added and frozen at -70°C.

[0075] 4. rETX m3 CSA M4△11 Expression and identification of

[0076] The BL / EC strain of Escherichia coli was inoculated into 3 mL of LB liquid culture medium containing kanamycin, and after shaking culture at 37°C for 4 hours, an IPTG solution with a final concentration of 0.5M was added for induction culture for 4 hours. After the bacterial culture was completed, the bacteria were collected by centrifugation, and 10 mL of lysis solution [0.02 mol / LTris buffer (pH 7.2), 0.3 mol / LNaCl] was added per gram of bacteria to resuspend the bacteria. The bacteria were ultrasonically disrupted in an ice water bath for 30 minutes. The disruption conditions were: working for 9 seconds, resting for 9 seconds, and the ultrasonic power was 400 W. The disrupted bacterial solution was centrifuged at 12000 r / min for 30 minutes at 4°C, the precipitate was discarded, and the supernatant was collected. Take 30 μL of the supernatant and add 10 μL of 4×SDS-PAGE loading buffer, act at 70°C for 10 minutes, and perform 12% SDS-PAGE electrophoresis. The results are shown in the table. Figure 1.from Figure 1 It can be seen that rETX m3 CSA M4△11 It is present in large quantities in the supernatant of bacterial lysate, expressed in a soluble form with good expression levels.

[0077] 5. rETX m3 CSA M4△11 Western blot identification

[0078] The fusion protein obtained in the above steps was identified by Western blot using anti-His antibody. Figure 2 .from Figure 2 It can be seen that the expression ratio of the fusion protein in the cell supernatant and the lysis precipitate is roughly the same. Since the fusion protein expressed in a soluble form in the cell lysis supernatant has a spatial structure that is closest to the wild-type toxin, we will purify the supernatant fusion protein.

[0079] 6. rETX m3 CSA M4△11 Purification of Escherichia coli BL / EC strain was inoculated into 1L LB liquid medium containing kanamycin for fermentation and cultured at 37℃ with shaking. 600 When the pH value is 1.2-1.6, add IPTG solution with a final concentration of 0.5M to induce the culture for 4 hours. After the bacterial culture is completed, the bacteria are collected by centrifugation, and the supernatant is collected according to the method in step 4. From the supernatant collected in the above step 4, the soluble expressed fusion protein in the supernatant of bacterial lysis is purified according to the instruction manual of the Ni-IDA affinity chromatography medium kit, and filtered through a 0.22μm pore size filter membrane to obtain the initially purified fusion protein.

[0080] 7. rETX m3 CSA M4△11 Determination of toxicity to mice This patent selects the method of tail vein injection to detect the toxicity of the fusion protein to mice in accordance with the provisions of the third volume of the "Pharmacopoeia of the People's Republic of China" (2015 edition) (China Veterinary Pharmacopoeia Committee, Veterinary Pharmacopoeia of the People's Republic of China, 2015 edition, Volume III, China Agriculture Press, 2016, hereinafter referred to as the "China Veterinary Pharmacopoeia"). At the same time, two negative controls, protein eluate and gastric liver enzyme digestion soup, and non-mutated recombinant ETX (rETX) and CSA (rCSA) were set as positive controls. The results showed that when the inoculation dose of the fusion protein was 100 μg, all mice were alive and had no adverse reactions, while rETX and rCSA could cause 5 / 5 deaths of mice when inoculated with 0.005 μg and 0.04688 μg, respectively. The results show that rETX m3 CSA M4△11 It was non-toxic in mice and was determined to be a non-toxic fusion protein.

[0081] Table 1 rETX m3 CSA M4△11 Toxicity to mice

[0082]

[0083] 8. rETX m3 CSA M4△11 Immunogenicity test: Conducted in accordance with the method specified in the Chinese Veterinary Pharmacopoeia.

[0084] (1) Serum neutralization method: Select 4 healthy rabbits weighing 1.5-2.0 kg and 4 sheep aged 6-12 months and weighing 30-40 kg, whose toxin neutralization titers to D-type Clostridium perfringens and Clostridium putrefaciens toxins are less than 1, that is, they are all antibody-negative, and inject 2.0 mL of the vaccine subcutaneously or intramuscularly into the neck of each. 21 days after vaccination, blood was collected, serum was separated, and 0.4 mL of each was mixed with D-type Clostridium perfringens toxin (containing 12 or more mouse MLD) and Clostridium putrefaciens (containing 4 or more mouse MLD), and then placed at 37°C for 40 minutes, and then injected intravenously into 2 mice weighing 16-18 g, 0.3 mL / mouse. At the same time, 2 mice from the same batch were injected with 1 MLD of the same toxin as the toxin serum mixture as a control. The mice tested for Clostridium putrefaciens were observed for 3 days, and the mice tested for D-type Clostridium perfringens were observed for 1 day, and the results were determined.

[0085] All the control mice died, and the serum neutralization titers against type D Clostridium perfringens and Clostridium putrefaciens toxins reached 3 or above (0.1 mL of immune animal serum neutralized 3 or more MLD toxins) and 1 or above (0.1 mL of immune animal serum neutralized 1 or more MLD toxins), respectively, and were judged to be qualified.

[0086] (2) Immunization challenge method: 12 healthy rabbits weighing 1.5-2.0 kg were divided into two groups, each with 6 rabbits. 4 rabbits in each group were injected with 2.0 mL of vaccine subcutaneously or intramuscularly in the neck. 21 days after vaccination, 1 MLD of D-type Clostridium perfringens and Clostridium putrefaciens toxin were injected intravenously, respectively, and observed for 3-5 days. All rabbits in the control group should die, and at least 3 rabbits in the immunization group should be protected.

[0087] The results were as follows Figure 4As shown, after one immunization, the toxin neutralizing antibody titer in rabbit serum reached 25-35 MLD (0.1 mL of immunized animal serum neutralized 25-35 MLD toxin) and 6-10 (0.1 mL of immunized animal serum neutralized 6-10 MLD toxin) for D-type Clostridium perfringens and Clostridium putrefaciens toxins, and the toxin neutralizing antibody titer in sheep serum reached 8-15 MLD (0.1 mL of immunized animal serum neutralized 8-15 MLD toxin) and 5-8 (0.1 mL of immunized animal serum neutralized 5-8 MLD toxin) for D-type Clostridium perfringens and Clostridium putrefaciens toxins. The results of the challenge showed that all rabbits in the control group died, and 100% of the rabbits in the immunized group were protected. The Chinese Veterinary Pharmacopoeia stipulates that the toxin antibody titer in rabbit serum against type D Clostridium perfringens and Clostridium putrefaciens toxins reaches 3 (0.1 mL of immunized animal serum neutralizes 3 MLD toxin) and 1 (0.1 mL of immunized animal serum neutralizes 1 MLD toxin), respectively, which is considered qualified. Therefore, the vaccine produced by this application has a serum neutralization titer of rabbits and sheep after one immunization that exceeds the provisions of the current Chinese Veterinary Pharmacopoeia, proving that the vaccine has good immunogenicity.

Claims

1. A non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine, characterized in that The vaccine contains a fusion protein of a mutant of Clostridium perfringens epsilon toxin (ETX) and a mutant of Clostridium putrefaciens alpha toxin (CSA) expressed by Escherichia coli; compared with the wild-type mature ETX, the 30th tyrosine mutates to alanine, the 106th histidine mutates to proline, and the 196th tyrosine mutates to alanine in the fusion protein; compared with the wild-type mature CSA, the 54th cysteine ​​mutates to leucine, the 264th asparagine mutates to alanine, the 269th histidine mutates to alanine, and the 310th tryptophan mutates to alanine in the fusion protein; the fusion protein simultaneously lacks 11 amino acids from positions 212 to 222; the strain used for vaccine preparation is the Escherichia coli expressing the fusion protein, which is named as the Escherichia coli BL / EC strain, with the deposit number: CGMCC No.17165; This vaccine is used to prevent sheep blight and enterotoxemia caused by infection with Clostridium perfringens and Clostridium putrefaciens.

2. The non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine as claimed in claim 1, characterized in that The fusion protein has a 6*His tag added to its C-terminus for easy protein purification.

3. The method for preparing the non-toxic Clostridium perfringens and Clostridium putrefaciens fusion protein vaccine as claimed in claim 1, characterized in that The Escherichia coli BL / EC strain expressing the fusion protein is used as a vaccine production strain, and after fermentation culture, induction expression, bacterial cell crushing, soluble antigen protein separation and purification, a biphasic oil adjuvant is added and mixed to prepare the vaccine.

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