CN114230645A

CN114230645A - Protective antigen protein of porcine toxigenic pasteurella multocida, application thereof and vaccine

Info

Publication number: CN114230645A
Application number: CN202210030958.XA
Authority: CN
Inventors: 牛旻; 杨瑞华; 李国强; 鲁璐; 肖红照; 姚菓; 贾宾; 程帅
Original assignee: Henan Xinghua Biotechnology Co ltd
Current assignee: Henan Xinghua Biotechnology Co ltd
Priority date: 2022-01-12
Filing date: 2022-01-12
Publication date: 2022-03-25

Abstract

The invention relates to the technical field of immunology, in particular to a protective antigen protein of porcine toxigenic pasteurella multocida, application thereof and a vaccine. The protective antigen protein is a polypeptide fragment of 1 st to 520 th of the N end of PMT protein. Experiments show that the protective antigen protein has higher immunogenicity and can effectively prevent and/or treat atrophic rhinitis of pigs. The invention also provides a PET-PMT-N strain of an escherichia coli expression system for expressing the antigen protein, 95% soluble protein can be obtained through expression and purification, the problems of purification and renaturation of inclusion body protein are avoided, the yield of target protein obtained through purification of PMT-N is improved to 0.4-0.8mg/ml, the production capacity is greatly improved, the vaccine has good immune effect and high safety, the preparation process is simple, inactivation is not needed, the operation is simple, and the production amplification is facilitated.

Description

Protective antigen protein of porcine toxigenic pasteurella multocida, application thereof and vaccine

Technical Field

The invention relates to the technical field of immunology, in particular to a protective antigen protein of porcine toxigenic pasteurella multocida, application thereof and a vaccine.

Background

The Atrophic Rhinitis (AR) of pig is a kind of chronic respiratory disease, which is mainly characterized by extensive expansion of nasal cavity and atrophy of mucous membrane, submucosa and bone tissue, has high contagious property, can reduce the resistance of swinery, deform face, slow growth speed, increase feed conversion ratio and increase input cost, increases the elimination rate of pig, and can seriously cause secondary infection to cause various diseases such as mycoplasma, meningitis or pig reproduction-respiratory tract syndrome, etc., thereby bringing great economic loss to the breeding industry of live pigs.

According to the etiology and the pathogenesis, the disease can be divided into Non-Progressive atrophic rhinitis (NPAR) and Progressive Atrophic Rhinitis (PAR). Non-progressive atrophic rhinitis is mainly caused by bordetella bronchiseptica, Bb is a gram-negative bacterium, and the causative toxin is Dermonecrotic toxin (DNT), which is an important, but not the only, factor that causes atrophic rhinitis. The single bordetella bronchiseptica infection is not serious, and the defect of the nasal bone can be regenerated.

Progressive atrophic rhinitis is mainly caused by toxigenic pasteurella multocida, which has various virulence factors including toxins, adhesins and pili. The nasal mucosa is easy to be infected by the secondary toxigenic Pasteurella multocida strain D after being damaged, and the Pasteurella Multocida Toxin (PMT) secreted by the infected toxigenic Pasteurella multocida strain D can enter cells to stimulate the cells of the interstitial tissue of the upper respiratory tract to be proliferated continuously. PMT toxin can induce cell synthesis and promote growth and division of bone cells, on one hand, the turbinate bone is absorbed due to massive proliferation of osteoclasts, and on the other hand, the regeneration of the turbinate bone is stopped due to inhibition of the function of the Gegen Bordetella cells, so that the shape and growth of the turbinate bone are abnormal.

With the stricter and stricter banning system of antibiotics in China, people have deeply understood the pathogenic mechanism of atrophic rhinitis of pigs and begin to attach importance to the effectiveness of AR control by AR vaccine. Currently, the commercial vaccines for prevention and control are mainly inactivated vaccines, toxoid vaccines, and subunit vaccines. Although the immunoeffect of the toxoid vaccine is superior to that of the inactivated vaccine, the PMT amount naturally secreted by the thallus is very limited, the artificial expression efficiency is low, the cost is high, the purification process is complex, and the problem of difficult large-scale production exists.

CN105797152A A vaccine composition and its preparation method and application introduce a subunit vaccine combining PRN-N, PRN-C, PMT-N and PMT-C4 protein, also through constructing recombinant Escherichia coli strain, express the above 4 proteins separately, and mix the protein and inactivate and prepare the above subunit vaccine, have better immune protection effect. The method needs to construct 4 recombinant strains, carries out 4-line amplification production to respectively express 4 proteins, directly mixes the 4 subunit proteins without purification and inactivates at 60 ℃ to combine the 4 subunit proteins into a vaccine, has high endotoxin content in the vaccine, influences the safety of the vaccine, easily causes swinery stress due to excessive impurity proteins in the vaccine without purification, has complicated operation, greatly increases the production cost of a production line, has the expression quantity of the 4 proteins of 212 plus materials per mu g/ml, has not ideal protein expression quantity, has low production capacity and is not beneficial to amplification production.

Disclosure of Invention

In view of the above, the present invention aims to overcome the defects of the prior art and provide a PMT-N protein with high safety and high immunogenicity.

The second purpose of the invention is to provide a recombinant strain with high yield of soluble PMT-N protein.

The third purpose of the invention is to provide a subunit vaccine for treating and/or preventing atrophic rhinitis of pigs.

The invention is realized by the following technical scheme:

a protective antigen protein of porcine toxigenic Pasteurella multocida is PMTN-terminal protein, and the amino acid sequence of the protective antigen protein is as follows, SED ID NO: 1 is shown.

Preferably, it is obtained by co-expression of a protein expression system; in some embodiments of the invention, the protein expression system is an E.coli expression system.

The invention also provides a preparation method of the protective antigen protein, which comprises the steps of connecting the nucleic acid molecule for coding the PMT N-terminal protein with an expression vector to construct a recombinant expression vector, transforming the recombinant expression vector into escherichia coli, culturing, and carrying out induced expression to obtain the protective antigen protein.

The invention also provides nucleic acid molecules encoding the protective antigenic proteins of the invention.

The invention also provides a biological material containing the nucleic acid molecule, wherein the biological material is an expression vector or a host bacterium or a host cell.

Preferably, the biological material is an expression vector, and the framework of the expression vector is a PET series vector.

Preferably, the biological material is a host bacterium; the host bacterium is escherichia coli; in some preferred embodiments, the escherichia coli is escherichia coli BL 21; in some embodiments, the biological material comprising the PMT-N terminal protein constructed according to the present invention is a PET-PMT-N recombinant bacterium.

Experiments prove that PMT-N end protein expressed by the PET-PMT-N recombinant bacteria constructed by the invention can be up to 95% soluble, the problems of inclusion body protein purification and renaturation are avoided, the yield of target protein obtained by purifying PMT-N can be improved to 0.4-0.8mg/ml, the production capacity is greatly improved, and animal experiments prove that the protein has better immunogenicity, high safety, no need of inactivation, simple operation and contribution to production amplification.

The invention also provides the application of the protective antigen protein, the nucleic acid molecule or the biological material in preparing the atrophic rhinitis vaccine of the pig.

The invention also provides a subunit vaccine for treating and/or preventing the atrophic rhinitis of pigs, which comprises the protective antigen protein and a vaccine adjuvant.

According to the invention, the PMT-N protein is connected with PET series carriers to transform escherichia coli, and the solubility of the target protein can be improved to 95% by using the recombinant strain PET29b-PMT-N obtained by immunogenicity comparison and PET series carrier screening, so that the soluble yield of the PMT-N target protein is improved to 0.4-0.8mg/ml, the problems of inclusion body protein purification and renaturation are avoided, and the efficacy test verifies that the PMT-N protein has good immunogenicity, and the production practicability is increased.

The invention constructs a series of recombinant bacteria containing different PMT-N-terminal proteins, and finally obtains the SED ID NO with higher immunogenicity: 1, PMT-N-terminal protein. The invention further constructs recombinant plasmids by enzyme digestion and enzyme linkage of the protein and different PET series carriers, induces, expresses and purifies the target protein, screens out the recombinant PET29b-PMT-N strain with the highest soluble expression quantity, and proves that the PMT-N protein prepared by the invention can be used for preparing vaccines for treating the atrophic rhinitis of pigs through a large amount of biological data, and has the advantages of high purity, high yield, no need of inactivation, simple operation and contribution to production and amplification.

The amino acid sequence provided by the invention is as follows: the protective antigen protein of the swine toxigenic pasteurella multocida shown in the 1 has high immunogenicity, and can effectively prevent and/or treat atrophic rhinitis of swine. After PMT-N protein expressed by the PET-PMT-N strain provided by the invention is purified by nickel column chromatography, the protein has high purity and good immunogenicity, and the batch treatment has low endotoxin content (below 3000 EU/ml), is safer, has high yield, does not need renaturation, does not need inactivation, and has lower cost.

The soluble yield of PMT-N protein expressed by the PET-PMT-N strain is 2-4 times higher than that of the common series strains with PET carriers, and the production and the amplification are convenient. The invention solves the following problems in the prior art:

1. solves the problems that the purification process of the naturally secreted PMT amount is complex, an inactivation process is required to be added, and the large-scale production is difficult;

2. the problems that most target proteins in the existing subunit vaccine are expressed by inclusion bodies, the inclusion body proteins have no biological activity and the renaturation process is complex to operate, or the soluble expression quantity of the target proteins in supernatant is low, the target proteins in thallus precipitates are directly discarded without being purified by the renaturation process to cause waste, the content of endotoxin in the inclusion body vaccine is high, the safety of the vaccine is poor and the like are solved;

3. solves the problem of poor safety and immunogenicity caused by low purity of target protein in the existing subunit vaccine.

Drawings

FIG. 1 shows the protein electrophoresis pattern of the expressed protein PMT-N.

Detailed Description

The invention provides a protective antigen protein of porcine toxigenic pasteurella multocida, application thereof and a vaccine. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless otherwise specified, the test materials used in the present invention are all common commercial products and are all available on the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 construction of recombinant strains

1.1 construction of expression engineering bacteria

According to literature investigations: the full length of PMT is toxic, the immunogenicity of PMT-N is better than that of PMT-C end, and then 3 PET-PMT full length (BL21), PET-PMT-N (BL21) and PET-PMT-C (BL21) expression engineering bacteria for expressing the full length of PMT, PMT-N (the segment of PMT N end from 1 st to 520 th site) and PMT-C protein (the segment of PMT from 520 th site to 1295 th site) are respectively constructed through vector screening.

1.2 PMT full Length, PMT-N, PMT-C protein expression, validation and purification quantitation

The full length of the expression engineering bacteria PET-PMT (BL21), PET-PMT-N (BL21) and PET-PMT-C (BL21) are respectively streaked on an LB (corresponding resistance) solid plate and cultured for 16h at 37 ℃, single colonies are respectively selected and inoculated in 5ml of LB liquid culture medium containing 100 mu g/ml corresponding resistance, shaking table (150r/min) at 37 ℃ is used for culturing for 16h, then the single colonies are inoculated in 500ml of LB liquid culture medium containing 100 mu g/ml corresponding resistance at 1 percent and cultured on shaking table (150r/min) at 37 ℃ until OD 0.6-0.8, 50mmol/L of isopropyl thiogalactoside (IPTG) is added, and the low-temperature induction is carried out overnight.

Carrying out centrifugation on the gene expression engineering bacteria PET-PMT full length (BL21), PET-PMT-N (BL21) and PET-PMT-C (BL21) in a centrifuge at 8000r for 10min to collect bacteria, and carrying out bacteria collection according to the bacteria: PBS 1:20 redissolving, 60% power over 5s stopping for 5s, ultrasonication for 30min, centrifuging for 30min at 10000r, filtering the supernatant through a 0.45 mu m filter membrane, purifying by nickel column, washing impurities by 100mmol/L imidazole +20mmol/L PB solution, eluting by 500mmol/L imidazole +20mmol/L PB solution, taking the bacterial redissolved sample in the process, crushing the supernatant and the precipitated sample, washing impurities and eluting by nickel column chromatography, adding SDS loading buffer solution, performing SDS polyacrylamide gel electrophoresis analysis, judging whether the protein is correct by the size of the protein band, determining the concentration of the eluted protein by BSA quantitative method, repeating the culture for 5 times, finally, after connecting with a PET carrier through an electrophoresis chart, determining that the solubility of the PMT full-length target protein in the supernatant is about 20%, and the solubility of the PMT-N target protein in the supernatant is about 95% (as shown in the protein electrophoresis chart of figure 1-N), the soluble ratio of PMT-C target protein in the supernatant is about 85%, and the soluble expression yield of PMT full-length protein is calculated to be 0.2-0.4mg/ml, the soluble expression yield of PMT-N protein is calculated to be 0.4-0.8mg/ml, and the soluble expression yield of PMT-C protein is calculated to be 0.3-0.5 mg/ml.

Example 2 vaccine preparation

2.1 preparation of PMT full-Length vaccine, PMT-N vaccine, PMT-C vaccine, PMT-N + PMT-C vaccine

Respectively taking a certain amount of PMT full-length protein (1), PMT-N protein (2) and PMT-C protein (3) prepared in example 1, adding a certain amount of whole Bordetella inactivated liquid into PMT-N + PMT-C protein (4), adding a certain proportion of aqueous adjuvant, uniformly stirring by using a magnetic stirrer, subpackaging, and obtaining the pig atrophic rhinitis subunit vaccine after inspection is qualified, wherein the antigen content in the vaccine is 50-200 mug/ml, and the ratio of the antigen to the adjuvant in the vaccine is as follows:

TABLE 1 compositions of vaccines 1, 2, 3, 4

Note: "-" indicates no addition.

Example 3 vaccine sterility test

Sterility test the 3 subunit vaccines were tested according to the appendix of the current "pharmacopoeia of Chinese beasts". All the results were aseptically grown.

Example 4 vaccine safety test

Selecting piglets of 20 days old to randomly divide into 8 groups, each group comprises 10 heads, respectively selecting the groups with the lowest concentration and the highest concentration of the vaccines 1, 2, 3 and 4 for immunization, injecting the vaccines into the neck, and observing the stress condition 14 days later by 2 ml/head.

The results show that vaccines 2-4 (including the highest and lowest concentration groups) have no apparent stress.

The lowest concentration group of pigs in the experimental result of the vaccine 1 has obvious stress (according with the result that PMT (PMT) has toxicity in the literature research), and the specific stress condition is as follows: sudden collapse, congestion and swelling of the canthus, dyspnea, even the symptoms of lying down, convulsion of limbs, trembling of the body and the like in the highest concentration group, and reduction in body temperature and increase in food consumption were not selected in the next experiment.

Example 5 vaccine efficacy test

Selecting 280 mice with 15-20g of pasteurella multocida PMT toxin antibody negative and pasteurella multocida antigen negative detected by PCR, randomly dividing into 14 groups, wherein each group comprises 20 mice, respectively immunizing the vaccines 2, 3, 4, PBS, the commercial vaccine 1, the commercial vaccine 2, the vaccine prepared by the PET28a-PMT-C (837-, recording the survival condition of the mice 7 days after the challenge, carrying out the challenge of bordetella on the other half, carrying out the challenge of the abdominal cavity, wherein each mouse is 0.4ml, the bacterium solution of the challenge contains bordetella 1x10^9CFU/ml, recording the survival condition of the mice 7 days after the challenge, and the statistical data are survival number and protection rate, and the experimental results are shown in the following table:

TABLE 2 statistics of survival after challenge pasteurisation

TABLE 3 statistics of survival after challenge Boehringer

As can be known from a safety test, the PMT has obvious toxicity in full length, and a potency test can judge that the whole bordetella has good protection on bordetella, the immune protection effect of the vaccine 2 after the challenge pasteurella is superior to that of the vaccine 3 and the vaccine 4, and the challenge protection effect on the bordetella is superior to that of the commercial vaccine 1 and the commercial vaccine 2 for the pernicious nose when the PMT-N protein antigen content is 200 mu g/ml, and is superior to that of the PET28a-PMT-C (837-. And the PMT-N protein (the polypeptide fragment at the 1 st-520 th site of the PMTN end) with better immunogenicity and high safety is determined.

Therefore, the vaccine composed of the PMT-N protein and the bordetella has good immunogenicity, high safety, no need of inactivation, high soluble yield of the PMT-N protein, no difficulty of inclusion body protein purification and renaturation, and convenient production and amplification.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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Claims

1. A protective antigen protein of porcine toxigenic Pasteurella multocida is a PMT N-terminal protein, and the amino acid sequence of the protein is as follows, SED ID NO: 1 is shown.

2. The protective antigenic protein of claim 1, which is co-expressed from a protein expression system.

3. The protective antigenic protein of claim 5, wherein said protein expression system is an E.

4. The method for preparing the protective antigenic protein of claim 1, wherein the protective antigenic protein is obtained by connecting a nucleic acid molecule encoding the PMTN-terminal protein with an expression vector to construct a recombinant expression vector, transforming the recombinant expression vector into escherichia coli, culturing, and inducing expression.

5. A nucleic acid molecule encoding a protective antigenic protein as claimed in any one of claims 1 to 3, having the sequence as defined by SED ID NO: 2, respectively.

6. A biological material comprising the nucleic acid molecule of claim 5, said biological material being an expression vector or a host.

7. The biomaterial according to claim 6, wherein the biomaterial is an expression vector, and the scaffold of the expression vector is a PET series vector.

8. Use of the protective antigenic protein of any one of claims 1 to 3, the nucleic acid molecule of claim 5, or the biomaterial of claim 6 or 7 for the preparation of a vaccine for atrophic rhinitis in pigs.

9. A subunit vaccine for treating and/or preventing atrophic rhinitis in pigs, comprising the protective antigenic protein of claim 1 and a vaccine adjuvant.