CN109078178B - Clostridium perfringens β toxin recombinant subunit vaccine and production method thereof - Google Patents

Clostridium perfringens β toxin recombinant subunit vaccine and production method thereof Download PDF

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CN109078178B
CN109078178B CN201810868174.8A CN201810868174A CN109078178B CN 109078178 B CN109078178 B CN 109078178B CN 201810868174 A CN201810868174 A CN 201810868174A CN 109078178 B CN109078178 B CN 109078178B
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杜吉革
陈小云
朱真
薛麒
刘莹
印春生
李启红
康凯
姚文生
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China Institute of Veterinary Drug Control
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Abstract

The invention relates to a clostridium perfringens β toxin recombinant subunit vaccine and a production method thereof, the clostridium perfringens β toxin recombinant subunit vaccine prepared by the invention is produced by adopting recombinant clostridium perfringens β toxin protein which is optimized by codons and contains 4 amino acid mutations, namely the integrity and the space conformation of natural toxin protein are kept to the maximum extent, thereby the immunogenicity of the vaccine is kept, and the biological potential safety hazard caused by single amino acid mutation is avoided.

Description

Clostridium perfringens β toxin recombinant subunit vaccine and production method thereof
Technical Field
The invention relates to a clostridium perfringens β toxin recombinant subunit vaccine and a production method thereof, belonging to the field of biological products for livestock.
Background
Clostridium perfringens, also called clostridium welchii, is an important disease of both human and livestock, and is one of the main pathogens of traumatic gas gangrene and human food poisoning as well as sheep plague, lamb dysentery, cattle and sheep necrotic enteritis and cattle and sheep enterotoxemia, causing huge economic loss to the animal husbandry.clostridium perfringens is classified into A, B, C, D, E five toxin types according to the species producing 4 main lethal exotoxins α, β, gamma and iota, and has the characteristics of acute onset, short course and extremely high mortality rate.
The commercial vaccines used at present are mainly inactivated vaccines, and although certain effects are achieved in the aspect of preventing clostridium perfringens disease of animals, the vaccines still expose 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 involves the inactivation of exotoxin, and has the biological potential safety hazards of toxin leakage or incomplete inactivation and the like; in addition, various microtoxins and bacterial metabolites in culture supernatant are often used as allergens of immunized animals, and the inoculated animals are easy to generate adverse reactions, so that the immune effect is reduced and even the immunity fails. Therefore, the development of the clostridium toxin genetic engineering vaccine with good safety, high effective antigen content and strong immunogenicity is the future development direction.
β toxin is the main pathogenic factor of C type perfringens bacterium, it has cytotoxicity and lethality, and can cause necrotic enteritis in human and animal, although the exogenous recombinant CPB molecule has a certain immune protection, but needs to consider the toxicity of protein.
In the prior art, the expression and purification method of main exotoxin proteins of clostridium perfringens is relatively complex, the expression products usually exist in the form of insoluble inclusion bodies, and the reports of soluble protein expression are very few at home and abroad. Since the expression product in inclusion bodies is biologically inactive, denaturation and renaturation treatments are required. The denaturation and renaturation of protein are a very complex process, the renaturation conditions of different proteins are different, and the renaturation rate is difficult to improve, which is a main restriction factor limiting the application of the protein. This problem is well overcome by using soluble expression. How to construct soluble expression vectors and optimize efficient expression methods of soluble proteins is a hot topic of research in the field for a long time.
The recombinant subunit vaccine of clostridium perfringens β toxin is produced by adopting the recombinant clostridium perfringens β toxin protein which is optimized by codons and contains 4 amino acid mutations, namely the integrity and the spatial conformation of the natural toxin protein are kept to the maximum extent, so that the immunogenicity of the vaccine is kept, and the biological potential safety hazard caused by single amino acid mutation is avoided.
Disclosure of Invention
The invention aims to prepare a recombinant subunit vaccine of clostridium perfringens β toxin for preventing diseases caused by clostridium perfringens type C infection.
Technical scheme of the invention
1. A recombinant subunit vaccine of clostridium perfringens β toxin is characterized in that the vaccine contains recombinant clostridium perfringens β toxin protein expressed by Escherichia coli, Escherichia coli of which the strain for vaccine preparation is recombinant expression clostridium perfringens β toxin protein is named as Escherichia coli (Escherichia coli) B L C2 strain, the strain is deposited by the common microorganism center of China Committee for culture Collection and management of microorganisms of national institute of microbiology, No. 3 of China academy of sciences, North West Lu 1 of south ench, Beijing, 01-18 days in 2018, and the number of the deposit is CGMCC No. 15238.
2. The clostridium perfringens β toxin recombinant subunit vaccine is characterized in that compared with a wild clostridium perfringens β toxin mature toxin, the recombinant clostridium perfringens β toxin protein contains 4 amino acid mutations, wherein arginine at the 212 th position is mutated into glutamic acid, leucine at the 268 th position is mutated into glycine, and tyrosine at the 266 th position and tryptophan at the 275 th position are mutated into alanine.
3. The recombinant subunit vaccine of clostridium perfringens β toxin is characterized in that the gene sequence of the recombinant clostridium perfringens β toxin protein is optimized by codon, and a malto glycoprotein (MBP) label is added at the N end, so that high-efficiency expression and soluble expression can be realized in escherichia coli more easily.
4. The recombinant subunit vaccine of clostridium perfringens β toxin is characterized in that the recombinant clostridium perfringens β toxin protein is a nontoxic mutant, so that the biological safety risk in the vaccine production process is greatly reduced.
5. The clostridium perfringens β toxin recombinant subunit vaccine is characterized in that the recombinant clostridium perfringens β toxin protein is soluble expressed in escherichia coli B L (DE3), so that the space conformation of the natural toxin protein can be retained to the maximum extent, the immunogenicity of the vaccine is kept, 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 vaccine are reduced.
6. The clostridium perfringens β toxin recombinant subunit vaccine is characterized in that the C end of the recombinant clostridium perfringens β toxin protein contains a 6 histidine (6His) tag, so that the purification of the protein is facilitated.
7. The preparation method of the clostridium perfringens β toxin recombinant subunit vaccine is characterized in that the escherichia coli B L C2 strain expressing clostridium perfringens β toxin recombinant protein is used as a vaccine production strain, and the vaccine is prepared by culturing, performing induced expression, breaking thalli, separating and purifying soluble antigen protein, and adding a biphasic oil emulsion adjuvant for mixing.
The invention mainly has the following advantages:
1. the β toxin is subjected to 4 amino acid mutations simultaneously for the first time, and the nontoxic recombinant β toxin is obtained, namely the integrity and spatial conformation of the natural toxin protein are retained to the maximum extent, so that the immunogenicity of the toxin is kept, and the biological potential safety hazard caused by single amino acid mutation is avoided.
2.β toxin is subjected to codon optimization, and high-efficiency expression in Escherichia coli is realized.
3. The β toxin and the solubilizing-aid label protein Maltoglycoprotein (MBP) are fused and expressed for the first time, so that the soluble expression in escherichia coli is realized, and the recombinant β toxin with excellent immunogenicity is obtained, 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 vaccine.
4. The subunit vaccine prepared by adopting the genetic engineering method replaces the traditional method of preparing toxin by culturing pathogenic clostridium and then inactivating and detoxifying, thereby greatly reducing the biosafety risk in the production process.
Detailed description of the invention
1. Preparation of recombinant subunit vaccine of clostridium perfringens β toxin
(1) Bacterial strain
The strain for preparing the vaccine is recombinant expression clostridium perfringens β toxin protein Escherichia coli (Escherichia coli) B L C2 strain, wherein the clostridium perfringens β toxin protein expressed by the strain contains 4 amino acid mutations (arginine at the 212 th position is mutated into glutamic acid, leucine at the 268 th position is mutated into glycine, tyrosine at the 266 th position and tryptophan at the 275 th position are mutated into alanine), an N end contains a malto glycoprotein (MBP) label, and a C end contains a 6 histidine (6His) label.
1) First order seed reproduction and identification
The freeze-dried strain is dissolved by a small amount of L B liquid culture medium, streaked and inoculated to a L B solid plate containing kanamycin, the plate is placed at 37 ℃ for culture for 12-16 hours, a single bacterial colony meeting the standard is selected, a L B liquid culture medium containing kanamycin is inoculated, the plate is placed at 37 ℃ for culture for 8-12 hours, the single bacterial colony and 50% of glycerol are mixed in equal proportion and then subpackaged, and the mixture is used as first-grade seeds for preparing seedlings after pure inspection and qualification.
2) And (3) propagation and identification of secondary seeds, namely taking the primary seeds, inoculating L B liquid culture medium containing kanamycin in an amount of 1%, and carrying out shake culture at 37 ℃ for 8-12 hours to obtain the secondary seeds.
(2) Antigen preparation for vaccine production
Inoculating 2% of qualified secondary seed into L B liquid culture medium containing kanamycin, culturing in fermenter, and culturing at 37 deg.C, pH 7.0 and dissolved oxygen above 20%600When the value is 10-15 ℃, the temperature is reduced to 15 ℃, and IPTG with the final concentration of 0.5 mmol/L is added for induced culture for 4 hours.
(3) Breaking bacteria
The thalli is collected by centrifugation, the thalli is resuspended according to the proportion that 10m L lysate (pH value 7.20.02 mol/L Tris buffer solution, 0.3 mol/L NaCl) is added to the wet weight of each gram of thalli, the thalli is broken by a high-pressure homogenizer at the pressure of 800bar, and the supernatant is collected by centrifugation.
(4) Purification of
Adding saturated ammonium sulfate into the collected supernatant to reach 30% saturation degree, fully mixing, standing for 4 hours at 2-8 ℃, centrifuging, collecting precipitate, re-suspending the precipitate by using buffer solution (pH value is 6.0, 0.01 mol/L phosphate buffer solution) with the same volume as the supernatant before ammonium sulfate precipitation, collecting supernatant after centrifugation, and filtering by using a 0.22 mu m pore size filter membrane.
(5) Protein content detection
Protein content was determined by BCA assay.
(6) Sterility testing
The method is carried out according to the appendix of the current Chinese veterinary pharmacopoeia (the Committee of the Chinese veterinary dictionary, the veterinary pharmacopoeia of the people's republic of China, the good quality of two or five years edition, the Chinese agricultural publishing agency, 2011, which is called the Chinese veterinary pharmacopoeia below). It should be grown aseptically.
(7) Vaccine formulation
Introducing a two-phase oil adjuvant (such as 201 adjuvant) into an oil phase tank, sterilizing at high pressure of at least 121 ℃ for 30 minutes, cooling to room temperature for later use, properly diluting purified protein qualified by inspection by PBS (pH value of 7.20.01 mol/L) according to the protein content measurement result, uniformly mixing, adding a water phase into an emulsification tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1(V/V), stirring for 20-30 min after the addition is finished, sampling for inspection, and subpackaging after the qualification.
2. Detection of recombinant subunit vaccine of clostridium perfringens β toxin
(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 sucked and dropped on the surface of clean cold water, and the vaccine should spread in a cloud state.
The stable suction vaccine 10m L is added into a centrifuge tube, centrifuged at 3000r/min for 15min without demulsification, and the water separated out from the tube bottom is not more than 0.5m L.
The viscosity is determined according to the appendix of the Chinese veterinary pharmacopoeia and is in accordance with the regulations.
(2) The loading inspection is carried out according to the appendix of Chinese animal pharmacopoeia, and the loading inspection is in accordance with the regulations.
(3) The sterility test is carried out according to the appendix of Chinese veterinary pharmacopoeia, and the growth should be carried out aseptically.
(4) 4 healthy rabbits with the weight of 1.5-2.0 kg are used for safety inspection, and each rabbit is injected with vaccine 4.0m L intramuscularly or subcutaneously, and the rabbits should be healthy and alive after being observed for 10 days.
(5) Efficacy test
1) A serum neutralization method comprises the steps of injecting 4 healthy rabbits with the weight of 1.5-2.0 kg into the neck of the rabbits at the subcutaneous or intramuscular level for 2.0M L, collecting blood after 14 days after inoculation, separating serum, simultaneously carrying out secondary immunization on the rabbits in the same dose and in the same way, collecting blood after 21 days after secondary immunization, separating serum, respectively mixing the serum of the 4 immunized rabbits of the primary immunization and the secondary immunization in the same amount, taking 0.4M L of the mixed serum and C-type clostridium perfringens toxin (containing 4 mice M L D), mixing, placing the mixture at 37 ℃ for 40min, then injecting 2 mice with the weight of 16-20 g and 0.3M L/mouse per group, injecting 1M L D of the same mouse and toxin serum mixture respectively as a contrast, observing for 1 day, and judging the result.
The control mice all die, and the serum neutralization titer reaches 1(0.1M L immune animal serum neutralizes 1M L D toxin) to the C type clostridium perfringens toxin, namely the control mice are qualified.
2) The immune toxicity attacking method comprises the steps of taking 8 healthy rabbits with the weight of 1.5-2.0 kg, injecting vaccine 2.0M L into the neck part subcutaneously or intramuscularly, taking the rest 4 healthy rabbits as control groups, taking 4 immune group rabbits and 2 control group rabbits 14 days after inoculation, respectively injecting C-type clostridium perfringens toxin of 1M L D into the vein, observing for 5 days, simultaneously carrying out secondary immunization on the rest 4 rabbits by the same dose and the same way, carrying out secondary immunization 21D after secondary immunization, taking the rest 4 immune group rabbits and 2 control group rabbits, respectively injecting C-type clostridium perfringens toxin of 1M L D into the vein, and observing for 5 days.
The control rabbits all die, and the immune animals are protected by at least 3 animals, namely, the control rabbits are judged to be qualified.
Drawings
FIG. 1 shows the SDS-PAGE identification of recombinant genetically engineered bacterium B L C2 strain, in which M is1:Protein marker；PC1:BSA(1μg)；PC2BSA (2. mu.g); NC, uninduced cell lysate; cell lysate induced at 1:15 ℃ for 16 h; 2: cell lysate induced at 37 ℃ for 4 h; NC (numerical control)1Non-induced cell lysis supernatant; NC (numerical control)2Inducing no cell lysis precipitation; cell lysis supernatant induced at 15 ℃ for 16 h; cell lysis precipitation induced at 4:15 ℃ for 16 h; 5:37 ℃ for 4hLysing the supernatant; 6, cell lysis precipitation induced at 37 ℃ for 4 h. The figure shows that the expression of the target protein in soluble expression is the highest in cell lysis supernatant induced by 16h at 15 ℃, and accounts for about 35% of the total expression amount of the target protein.
FIG. 2 shows the result of Western blot (with anti-His antibody) identification of recombinant strain B L C2, wherein M2 is Western blot marker, cell lysate induced at 1:15 ℃ for 16h, cell lysate induced at 2:37 ℃ for 4h, cell lysis supernatant induced at 3:15 ℃ for 16h, cell lysis precipitate induced at 4:15 ℃ for 16h, cell lysis supernatant induced at 5:37 ℃ for 4h, and cell lysis precipitate induced at 6:37 ℃ for 4 h.
FIG. 3: western blot (adopting anti-C type clostridium perfringens toxin serum) identification result of the target protein, wherein M2 is Western blot marker; BSA; 2, the purified target protein.
The invention relates to biomaterial resource information
The invention relates to a microorganism which is Escherichia coli B L C2 strain of expression clostridium perfringens β toxin containing 4 amino acid mutations (arginine at 212 is mutated into glutamic acid, leucine at 268 is mutated into glycine, tyrosine at 266 and tryptophan at 275 are mutated into alanine), a malto-glycoprotein (MBP) label is contained at the N end, and a 6 histidine (6His) label is contained at the C end, the strain is delivered to China general microbiological culture collection management committee general microbiological center of China institute of microbiology, national institute of south Asian province No.1, 3, Beijing city on 01-18 days in 2018, and the collection number is CGMCC No. 15238.
Positive significance of the invention
The invention relates to a clostridium perfringens β toxin recombinant subunit vaccine and a production method thereof, wherein arginine at the 212 th position of mature toxin of wild clostridium perfringens β is mutated into glutamic acid, leucine at the 268 th position is mutated into glycine, tyrosine at the 266 th position and tryptophan at the 275 th position are mutated into alanine to obtain a β toxin mutant (B L C2 strain) which is nontoxic to animals.
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 identification of Escherichia coli B L C2 strain (also called clostridium perfringens β toxin 4 amino acid mutant in the invention)
1. Gene synthesis
According to the sequence of a natural protein gene of clostridium perfringens β toxin, 4 amino acid mutations are designed after codon optimization, arginine at the 212 th site of mature toxin of wild clostridium perfringens β toxin is mutated into glutamic acid, leucine at the 268 th site is mutated into glycine, tyrosine at the 266 th site and tryptophan at the 275 th site are mutated into alanine, so that a β toxin mutant which is nontoxic to animals is obtainedm4And contains 2097 nucleotides in total.
The specific nucleic acid sequence is shown as SEQ ID No.1,
Figure GDA0002170805750000071
Figure GDA0002170805750000081
the amino acid sequence is shown in SEQ ID No. 2.
Figure GDA0002170805750000082
Figure GDA0002170805750000091
2. Construction of fusion expression vectors
Artificially synthesized GMBPCPBm4The gene is used as a template, and PCR amplification is carried out by adopting a primer pair 1F/1R (sequence 3/sequence 4).
Wherein the sequence of the upstream primer 1F is as follows:
5'-ccgtctagag gtaccaaaac tgaag-3' 25 (SEQ ID NO: 3), wherein a restriction enzyme Xba I site and a protective base are introduced into the 5 ' end of the DNA sequence;
the sequence of the downstream primer 1R is as follows:
5'-ccgctcgagt tagtggtgat gatg-3' 24 (SEQ ID NO: 4), wherein a restriction enzyme XhoI site and a protective base are introduced into the 5 ' end of the DNA.
The PCR system comprises 10 × EX Taq Buffer 5 mu L4 mu L, upstream and downstream primers 2 mu L Taq enzyme 0.5 mu L respectively, full-length gene DNA template 2 mu L and ddH supplement2O to 50 mu L system, PCR reaction conditions are pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 160s, 33 cycles, and extension at 72 ℃ for 10 min.
Recovering the amplified target DNA band, digesting with Xba I/Xho I double enzyme, and connecting with pET28a digested with the same enzyme to obtain the inserted GMBP-CPBm4Gene positive clone pET28a-mMBPCPBm4The ligated plasmid was transformed into DH5 α competent cells, and a single clone was picked up into liquid culture L B containing kanamycinIn the medium, the mixture was cultured overnight with shaking at 37 ℃ and the plasmid was extracted for use.
3. Construction of genetic engineering strain for recombinant expression of clostridium perfringens β toxin mutant gene
The extracted plasmid is transformed into competent cells of Escherichia coli B L21 (DE3), a single clone is picked into a L B liquid culture medium containing kanamycin, the culture is performed overnight under shaking at 37 ℃, after PCR identification, a target DNA fragment is contained, the strain is named as Escherichia coli (E.coli) B L C2, 50% of glycerol L B with the same volume is added, the strain is frozen and stored at 70 ℃, and the strain is delivered to China general microbiological culture collection center of China microbiological culture collection administration of institute of microbiology, China institute of sciences, North West Lu No.1, 3, of the Yangyang region in Beijing city on 18 days in 2018, wherein the storage number is CGMCC No. 15238.
4. Expression of the protein of interest
The recombinant Escherichia coli (E.coli) B L C2 strain is inoculated in L B liquid culture medium containing 100m L kanamycin, after shaking culture at 37 ℃ for 4h, the temperature is reduced to 15 ℃, IPTG solution with the final concentration of 0.5 mmol/L is added to induce culture for 16h, after bacterial liquid culture is completed, the bacterial liquid is centrifugally collected, 10m L lysate [0.02 mol/L Tris buffer (pH value 7.2) and 0.3 mol/L NaCl ] is added according to the weight of each gram of bacterial body, the bacterial liquid is ultrasonically crushed in ice water bath for 30min under the conditions of working 9s, intermittent 9s and ultrasonic power of 400W, the crushed bacterial liquid is placed at 4 ℃, centrifuged for 10min at 12000r/min, supernatant is collected, 30 mu 5 supernatant is added into 4 × -PAGE sample buffer of 10 mu L, the supernatant is acted at 70 ℃ for 10min, 12% SDS-electrophoresis is carried out, and the result is shown in figure 1, a large amount of SDS-PAGE protein exists in the supernatant of 10 mu L, the target protein, and the optimal expression amount of the supernatant is induced expression under the conditions of 16h, 25 g induction expression is shown in SDS-PAGE.
5. Identification of the protein of interest
Western blot identification is carried out on the target protein by adopting target protein expression products under different induction conditions in the steps and adopting a murine anti-His monoclonal antibody (Sigma, cat # SAB2702219), and the result is shown in figure 2. As can be seen from FIG. 2, the expression level of the target protein was highest in the supernatant of cell lysis induced at 15 ℃ for 16 hours, and was about 40% of the total expression level of the target protein. The spatial structure is closest to that of wild-type toxin due to the soluble expression of target protein in 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.
6. Purification of proteins of interest
And (3) adding saturated ammonium sulfate into the supernatant collected in the step (4) to 30% of saturation degree, fully mixing, standing for 4 hours at the temperature of 2-8 ℃, centrifuging, collecting precipitates, re-suspending the precipitates by using a buffer solution (0.01 mol/L phosphate buffer solution (pH value of 6.0)) with the same volume as that of the supernatant before ammonium sulfate precipitation, centrifuging, collecting the supernatant, and filtering by using a filter membrane with the aperture of 0.22 mu m to obtain the preliminarily purified target protein.
7. Reaction of protein of interest with antiserum raised against clostridium perfringens type C toxin
Western blot identification is carried out on the target protein by adopting the target protein purified in the steps and antiserum for resisting C-type clostridium perfringens toxin, and the result is shown in figure 3. As can be seen in FIG. 3, the fusion protein was reactive with antisera raised against Clostridium perfringens type C toxin.
Example 2
Toxicity test of nontoxic mutant of clostridium perfringens β toxin to mice
The toxicity of the nontoxic mutant of the clostridium perfringens β toxin to mice is determined to verify the actual attenuation effect of the mutant in vivo, and the purified clostridium perfringens β toxin 4 amino acid mutant recombinant protein mMBPCPBm4And C-type clostridium perfringens culture supernatants are respectively inoculated to 16-18 g of mice through tail veins at different dosages, 5 mice are injected in each dosage, 0.2m L/mouse, and as a result, when the inoculation dosage is 0.1mg, all the mice are healthy and alive without adverse reaction, and a wild type control group can cause the death of the mice 5/5 when being inoculated with 0.001m L.
TABLE 1 recombinant protein mMBPCPBm4Virulence in mice
Figure GDA0002170805750000111
Example 3
Immunogenicity test of Clostridium perfringens β toxin 4 amino acid mutant
1. Bacterial liquid culture
Inoculating 2% of culture solution of Escherichia coli genetic engineering bacteria B L C2 strain of Clostridium perfringens β toxin protein into L B liquid culture medium containing kanamycin, culturing in a fermentation tank, setting culture parameters of culture temperature 37 ℃, pH value 7.0 and dissolved oxygen higher than 20%, and culturing at OD600When the value is 10-15 ℃, the temperature is reduced to 15 ℃, and IPTG with the final concentration of 0.5 mmol/L is added for induction culture for 16 hours.
2. Breaking bacteria
The thalli is collected by centrifugation, the thalli is resuspended according to the proportion that 10m L lysate (0.02 mol/L Tris buffer solution (pH value 7.2) and 0.3 mol/L NaCl) is added into the body temperature of each gram of thalli, the thalli is broken by a high-pressure homogenizer at the pressure of 800bar, and the supernatant is collected by centrifugation.
3. Purification of
Adding saturated ammonium sulfate into the collected supernatant to reach 30% saturation degree, fully mixing, standing for 4 hours at 2-8 ℃, centrifuging, collecting precipitate, re-suspending the precipitate by using a buffer solution (pH value is 6.0, 0.01 mol/L phosphate buffer solution) with the same volume as the supernatant before ammonium sulfate precipitation, collecting the supernatant after centrifugation, and filtering by using a 0.22 mu m pore size filter membrane.
4. Protein content detection
Protein content was measured by BCA assay, resulting in a protein content of 400. mu.g/m L.
5. Sterility testing
According to the appendix of the Chinese animal pharmacopoeia. All the results were aseptically grown.
6. Vaccine formulation
Introducing a two-phase oil adjuvant (201 adjuvant) into an oil phase tank, sterilizing at the high pressure of at least 121 ℃ for 30 minutes, cooling to room temperature for later use, diluting purified protein qualified by inspection to the final concentration of 100 mu g/m L by PBS (pH value of 7.20.01 mol/L) according to the protein content measurement result, uniformly mixing, adding a water phase into an emulsification tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1(V/V), stirring for 20-30 min after the addition is finished, sampling for inspection, and subpackaging after the qualification.
7. Immunogenicity assays
The method is carried out according to the method specified in Chinese veterinary pharmacopoeia, and the specific test method comprises the following steps of injecting 4 healthy rabbits with the weight of 1.5-2.0 kg into the neck of each rabbit at the time of subcutaneous or intramuscular injection of vaccine 2.0m L, collecting blood and separating serum 14 days after inoculation, simultaneously carrying out secondary immunization on the rabbits in the same dose and in the same way, collecting blood and separating serum 21 days after secondary immunization, and respectively measuring the neutralizing titer of toxin antibodies in the rabbit serum after primary immunization and secondary immunization by adopting the following method:
the method comprises the steps of mixing the sera of 4 immunized rabbits in equal amount, taking 0.4M L of the mixed sera and C-type clostridium perfringens toxin (containing 4 mice M L D), mixing the sera and acting at 37 ℃ for 40min, then carrying out intravenous injection on 2 mice (16-20 g) and 0.3M L/mouse, simultaneously respectively using 2 mice in the same batch, respectively injecting 1M L D and toxin which is the same as the mixture of the toxin sera as a control, observing for 1 day, judging the result, if the control mice are completely dead, enabling the serum neutralization titer to reach 1 for the C-type clostridium perfringens toxin (neutralizing 1M L D toxin in the sera of 0.1M L immunized animals), and judging the mice to be qualified.
Through determination, after the primary immunization, the toxin antibody titer in the rabbit serum is 3 (namely 0.1M L rabbit serum can neutralize 3M L D C type clostridium perfringens toxins), and after the secondary immunization, the toxin antibody titer in the rabbit serum is 6 (namely 0.1M L rabbit serum can neutralize 6M L D C type clostridium perfringens toxins).
The results of the immunization challenge showed that after 14D of first immunization and 21D of second immunization, the animals of the immunized group were protected by 100% (4/4) and the animals of the control group were all dead by the otic intravenous injection of 1M L D Clostridium perfringens toxin C.
The titer of toxin antibodies in the serum of the domestic rabbit reaches 1 to the C-type clostridium perfringens toxin, and in an immune attack method, at least 3 immune animals are protected, so that the part of the C-type clostridium perfringens toxin in the vaccine is judged to be qualified.
In view of the fact that the existing commercial clostridial toxin vaccine in China needs to be inactivated and detoxified by formaldehyde, the potential biological safety hazard exists, and the safety of the vaccine in field use is also influenced; meanwhile, the existing commercial vaccine has the problem of unstable virus production in the production process, which causes unstable vaccine efficacy. Therefore, the vaccine produced by the application is an ideal candidate vaccine for upgrading and updating the current C-type clostridium perfringens toxin vaccine in China.
Figure IDA0001842944030000011
Figure IDA0001842944030000021

Claims (2)

1. A recombinant subunit vaccine of clostridium perfringens β toxin, characterized in that the vaccine contains recombinant clostridium perfringens β toxin protein expressed by escherichia coli;
compared with a wild clostridium perfringens β toxin mature toxin, the recombinant clostridium perfringens β toxin protein contains 4 amino acid mutation points, wherein arginine at the 212 th position is mutated into glutamic acid, leucine at the 268 th position is mutated into glycine, tyrosine at the 266 th position and tryptophan at the 275 th position are mutated into alanine, and a Maltoglycoprotein (MBP) label is added at the N end, wherein the C end of the recombinant clostridium perfringens β toxin protein contains a 6 histidine (6 × His) label;
the recombinant clostridium perfringens β toxin protein is a nontoxic mutant;
the vaccine strain for preparing the vaccine is Escherichia coli which recombinantly expresses clostridium perfringens β toxin protein and is named as Escherichia coli (Escherichia coli) B L C2 strain, the strain is preserved by the common microorganism center of the China Committee for culture Collection of microorganisms of China institute of microbiology, No. 3, Ministry of sciences, North Asian province, No.1, of Beijing city in 2018, and the preservation number is CGMCC No.15238 on 18 days 01.
2. The recombinant subunit vaccine of clostridium perfringens β toxin according to claim 1, which is prepared by using the escherichia coli B L C2 strain expressing clostridium perfringens β toxin recombinant protein as a vaccine production strain, culturing, performing induced expression, breaking thallus, separating and purifying soluble antigen protein, and adding a biphasic oil adjuvant for mixing.
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