CN102380095A - FMD trivalence polypeptide vaccine and preparation method and application thereof - Google Patents

Abstract

A kind of foot-and-mouth disease trivalent polypeptide vaccine and its preparation method and application, select O, Asia I, A type three kinds of foot-and-mouth disease viruses, take VP1 B cell determinant, and VP4 T-cell helper 15 amino acid fragments, concatenation, clone, not Containing carrier protein, construct a genetically engineered bacterium, undergo high-density fermentation, cell disruption, inclusion body renaturation, fusion protein separation and purification, freeze-drying to obtain antigenic protein products, and adjuvant homogenate to form a trivalent polypeptide vaccine. The gene recombinant trivalent polypeptide vaccine of the present invention contains 2 ^n-1 consecutive polypeptides encoded by the nucleic acid sequence shown in SEQ ID, wherein n is an integer of 1-5. The vaccine of the invention has good safety, high immunization titer, can be immunized once within half a year, is suitable for large-scale production, is convenient for storage and transportation, can effectively prevent and treat three subtypes of foot-and-mouth disease, and does not produce non-structural protein 3A.B of foot-and-mouth disease virus ., easy to distinguish from infected animals.

Description

A kind of foot-and-mouth disease trivalent polypeptide vaccine and its preparation method and application

technical field

The invention relates to a novel foot-and-mouth disease vaccine, in particular to a trivalent polypeptide vaccine produced by a genetic engineering method and its preparation method and application.

Background technique

The continuous expansion of domestic breeding scale will become the source of continuous growth for the animal vaccine industry. At present, my country's meat and egg production ranks first in the world, and it is the world's largest producer of livestock products. In 2004, the annual output value of my country's animal husbandry exceeded 1 trillion yuan for the first time, and the proportion of the output value of animal husbandry to the total agricultural output value was gradually increasing. Due to the continuous epidemics worldwide, foot-and-mouth disease, bird flu, mad cow disease, etc. have brought challenges to the prevention and control of animal diseases in countries all over the world. Foot and mouth disease (FMD) is an acute, febrile and highly contagious infectious disease of artiodactyls (pigs, cattle, sheep and camels) in animal husbandry. So far, all countries in the world except North America and Australia have experienced large-scale epidemics of foot-and-mouth disease. The prevalence of this disease has affected Germany (1937-1938), Europe (1951-1952), Turkey (1964-1965), Britain (1967-1968), Austria (1973), France (1974), South Korea (2002), etc. The country caused huge economic losses.

The clinical diagnosis of foot-and-mouth disease is characterized by blisters and ulcers on the oral mucosa, hooves and breast skin. It is caused by foot-and-mouth disease virus. It is extremely harmful to animal husbandry, the breeding of pigs, cattle and sheep, and the trade of livestock products. Foot and mouth disease virus (Foot and mouth disease virus, FMDV) is a small RNA virus. It has many transmission routes to pigs, cattle and sheep, and is highly virulent. The amount of detoxification of a sick cow can infect 1 million cows, and 1 gram of sick pigs has blisters The skin can make 100,000 pigs infected. It is easy to spread, spreads rapidly, has a wide range of epidemics, declines in production performance, high cost of control, morbidity is 100%, and the mortality rate of adult animals is generally 1-2%, but that of young animals is as high as 50%, or even 100%. Often several provinces, several continents or even the whole world happen at the same time. Both direct contact and indirect contact can be transmitted, mainly through the digestive tract, and can also be transmitted through the respiratory tract. Often cattle, sheep, and pigs become ill at the same time, and have the ability to infect multiple animals and have multiple antigen forms. Leaping transmission can occur throughout the year, and it is popular every 1-2 years or 3-5 years. In recent years, the occurrence of pigs has a tendency to expand. FMDV first replicates in the pharynx, then infects adjacent lymph nodes and enters the bloodstream, where it spreads to various organs and tissues. In most cases, animals develop clinical symptoms 2-14 days after infection. Older animals rarely die after infection with FMDV, but the animals' fertility, growth, and health are greatly affected. The International Office of Epizootics classifies FMDV as a severe infectious disease of Class A. Once FMDV occurs, it is difficult to control. After each outbreak, the infected livestock can only be slaughtered and collectively burned to avoid future troubles. Because foot-and-mouth disease spreads rapidly, is difficult to control, and has few remedial measures, it is called the "number one killer" of animal husbandry, and it is a devastating blow to animal husbandry. According to the regulations of the World Organization for Animal Health, once a foot-and-mouth disease outbreak occurs, the country concerned will automatically lose its qualification as a non-foot-and-mouth disease area.

Foot-and-mouth disease virus has significant antigenic diversity. Currently, there are 7 serotypes of FMDV found: O, A, C, SAT1, SAT2, SAT3 (namely South Africa 1, 2, 3) and Asia1 (Asia 1). Each serotype can further be divided into multiple subtypes. China was originally an O-type FMD endemic area, and neighboring countries were A-type FMD endemic areas. In the past 2 years, A, O, and Asia I types have been reported to be prevalent. FMD virus particles contain a positive-strand polarity single-stranded RNA consisting of 8500 nucleotides. The envelope of virus particles includes four structural proteins VP1 (molecular weight 34,000), VP2 (molecular weight 30,000), VP3 (molecular weight 26,000), and VP4 (molecular weight 13,500) surrounding single-stranded RNA. Sixty molecules of each of the four structural proteins constitute an icosahedral virion. The vertices of the icosahedron consist of the 141-160 region and the 200-213 region of the immunodeterminant region of the structural protein VP1. For FMD, vaccination is currently the most effective means of preventing FMD. Immunization vaccines are:

1. Attenuated vaccines and inactivated vaccines:

Traditional attenuated vaccines and inactivated vaccines are prepared by cultivating a large number of animal cells, then infecting the foot-and-mouth disease virus, cultivating, separating the virus, and inactivating the virus with chemical reagents. French company Merieux is the largest manufacturer of inactivated FMDV vaccine. Suspension cells and monolayer cell culture have low concentration, about 1*10 ⁶ , and the cost is high, and the preparation needs strict isolation and production under closed conditions; in addition, the storage and transportation of the vaccine requires cold chain, which increases the cost; the efficacy of inactivated FMDV vaccine The price of the vaccine varies greatly, and there are traces of re-strength and residual live viruses in the vaccine, which may cause epidemics and large-scale outbreaks; in addition, the quality of the vaccine often affects the milk secretion of dairy cows; although the use of inactivated FMDV vaccines does not often occur Anaphylactic shock or coma, but once it occurs, it is enough to cause serious consequences; finally, the first immunization should be given twice within 2 weeks to 2 months, and then injected every 6 months. The workload of immunization prevention is very large. Internationally, it is considered that animals immunized with inactivated FMDV vaccines are carriers of the virus (the impact of residual live virus particles after inactivation), and thus such animals and livestock products cannot enter the international trade market. Countries without FMDV, such as North America and Australia, do not produce inactivated vaccines locally, and strictly prevent the import of animals and livestock products from countries in epidemic areas.

2. Empty virus particle vaccines:

Although the FMDV empty shell virus-like particle vaccine has no danger of the spread of live virus, it is safe and effective, but it needs to be cultured by insect cells. Insect cell culture is divided into three types: mass suspension culture, microcarrier culture and silkworm larvae culture. The mass culture technology of the former two is difficult, the cost is high, the yield is low, and the price is high. The insect cell culture is lower than BHK-21 cells, and the latter Silkworm larvae need land to plant mulberry trees, and are limited by seasons, once a year.

3. Peptide vaccine:

Synthetic peptide vaccines can overcome the shortcomings of conventional vaccines, and have long been considered as the ultimate vaccines for the prevention of animal infectious diseases. In 1982, a synthetic polypeptide vaccine was developed. Using Escherichia coli as the host cell requires only 20 hours of fermentation and culture, which is much shorter than using animal cells to produce inactivated vaccines. Genetic engineering vaccines do not pollute the environment, have no risk of leakage, and are very safe. No refrigeration is required during storage, transportation and use, and the potency is stable. Moreover, the current FMDV vaccine, whether it is an inactivated FMDV vaccine or a peptide vaccine, cannot distinguish between immunized animals and infected animals through serological experiments, and confusion between immunized animals and infected animals will cause great damage to the market. Influence. Therefore, it is our constant goal to prepare a genetically engineered multivalent foot-and-mouth disease vaccine that has improved immune titer and can be used to distinguish immunized animals from infected animals.

For more than 20 years, the research on peptide vaccines has mostly focused on the single immune determinant of B cells of the VP1 coat protein. Further research has shown that the immune determinant of the structural protein VP1 is the 141-160 region and the 200-213 region. The polypeptide fragments of these two regions are related to The antibody produced after the carrier protein is chemically linked can neutralize the virion (Nature, 1982,298:30-33; J.Virology, 2000,74:4902-4907; J.Virology, 2001,75:3164-3174; J.Virology, 2001,75:3164-3174; . Virology, 2003, 77: 8633-8640; Vaccine, 2002, 20: 2603-2610). However, due to the low production of neutralizing antibodies, the immune protection effect of synthetic peptide vaccines after immunizing animals is not as ideal as people originally thought, and the value of use has even been denied. In the process of inducing immunity in the body, a single neutralizing epitope is not enough.

Studies in recent years have proved that in addition to the B cell immune determinant of VP1, the T cell immune cluster of VP4 needs to act synergistically to obtain a good protective effect of immune neutralizing antibodies.

The Asia1 genetic engineering vaccine developed by Fudan University and other units contains T-helper and VP1 immunodeterminant vaccines, which have a protective effect and can produce neutralizing antibodies.

The peptide vaccine synthesized by UBI Company in the United States has T-cell Helper polypeptide and VP1 B cell immunodeterminant polypeptide, which greatly enhances the effect of neutralizing antibodies (about 10 times), and has a clear immune protection effect. It has good safety: no fever symptoms, no allergic reactions, no malabsorption at the injection site, no impact on the health status and pregnancy of pregnant sows, and no impact on litter performance.

At present, there is a need for large-scale and low-cost production of peptide vaccines. However, chemically synthesizing peptide chains of more than 20 amino acids is very expensive. Therefore, it is necessary to develop biotechnology to mass-produce polypeptides through genetic engineering. However, practical applications are still far away. According to research reports on peptides in the past 20 years, except for insulin, genetic engineering has no advantage over chemical synthesis. The fundamental reason is that the theoretical development and practical application of genetic engineering have yet to be developed. Moreover, the rapid degradation of polypeptides in vivo is not conducive to the induction of corresponding antibodies. The FMDV polypeptide vaccine should be prepared by genetic engineering technology. Like other genetically engineered peptide products, production needs to solve a series of upstream design and downstream process problems. Although there are many reports, it is far from being applied.

Contents of the invention

The purpose of the invention is to overcome the defects of the prior art and provide a novel genetically engineered trivalent foot-and-mouth disease polypeptide vaccine. A kind of foot-and-mouth disease virus trivalent polypeptide vaccine of the present invention is prepared by a genetically engineered bacterium. There are four kinds of foot-and-mouth disease virus coat proteins, namely VP1, VP2, VP3, and VP4. -The DNA sequence of the 15-amino acid fragment of the cell helper linked to the immunogenic immunodeterminant region of the O-type VP1 protein was linked to the DNA sequence linked to the immunogenic immunogenic determinant region of the Asia I type VP1 protein A The DNA sequence of the immunogenic immunodeterminant region in the type VP1 protein is used as a repeating fragment, and the foot-and-mouth disease virus trivalent polypeptide vaccine contains 2 ^n-1 proteins encoded by the nucleic acid sequence shown in the sequence of SEQ ID, Wherein n is an integer of 1-5.

Another object of the present invention is to provide a method for constructing the foot-and-mouth disease vaccine.

In the trivalent polypeptide vaccine of the present invention, the genetically engineered bacterium contains the tandem genes and expresses them with the Lac plasmid carried in Escherichia coli (E. Coli). The vaccine also includes pharmaceutically acceptable carriers, auxiliary materials and/or immune adjuvants.

On the other hand, the present invention also provides a genetic engineering strain capable of expressing the foot-and-mouth disease polypeptide vaccine.

The plasmids carried by the genetic engineering strain respectively contain 2 ^n-1 nucleic acid sequences shown in concatenated SEQ ID, wherein n is an integer of 1-5.

The invention provides the use of the foot-and-mouth disease vaccine in preventing and treating the foot-and-mouth disease and the use in distinguishing infected animals from immunized animals.

The present invention also provides a preparation method of foot-and-mouth disease virus polypeptide vaccine of artiodactyl livestock, comprising gene recombination and preparation of recombinant fusion protein, characterized in that:

(1) For the synthesis of genetically engineered bacteria, the DNA sequence of the 15 amino acid fragments encoding the T-cell helper of VP4 was synthesized by chemical synthesis, and the DNA sequence of the immunogenic immunodeterminant region in the O-type VP1 protein was linked to Asia The DNA sequence of the immunogenic immunodeterminant region in the type I VP1 protein is connected with the DNA sequence of the immunogenic immunodeterminant region in the A type VP1 protein as an A/Asia I repeat fragment, which is connected in series 4 times;

(2) For synthetic genetically engineered bacteria, insert 4 repeating fragments into the Lac plasmid vector;

(3) Transforming the above-mentioned recombinant plasmid into Escherichia coli for expression to obtain a bacterial strain;

(4) Ferment the bacterial strain in nutrient-rich LB medium, the fermentation temperature is 37°C, and the fermentation time is 12-24 hours. Add ampicillin to the fermentation broth to make the final concentration 50 μg/ml, and IPTG, the concentration is 0.5 mM, centrifuged after fermentation to obtain bacterial cells, crushed cells, renaturation of inclusion bodies, separation and purification of fusion protein, freeze-dried to obtain the product, and homogenized with adjuvant to form a trivalent polypeptide vaccine.

The genetic engineering research on the polypeptide of the present invention includes the establishment of an upstream high-efficiency expression design and an intensive high-tech downstream process, and the establishment of a high-yield, low-cost simplified process. After the genes are concatenated and expressed in Escherichia coli, a trivalent polypeptide vaccine with ideal immunogenicity is obtained, and good results are obtained in immunization experiments with antibodies. Because the four times of expression in series are macromolecules, the amount of neutralizing antibodies produced is significantly increased, and the half-life of the polypeptide in vivo is also prolonged. The vaccine prepared by the polypeptide of the present invention can prevent and treat three types of foot-and-mouth disease O, A and Asia I. Foot-and-mouth disease trivalent genetically engineered peptide vaccine is one of a series of peptide drugs, which can be mass-produced cheaply. It only takes 12 hours to ferment and cultivate Escherichia coli as the engineered bacteria, and the genetically engineered vaccine has no pollution to the environment and no risk of leakage. The produced vaccine does not require a cold chain during storage, transportation and use. One immunization covers all subtypes in my country. Our genetically engineered foot-and-mouth disease vaccine can distinguish between immunized animals and infected animals in addition to high immune titer.

Using the vaccine of the present invention has the following advantages: 1) good safety, the present invention is a product of genetic engineering, not an inactivated vaccine, and there is no risk of disease outbreak caused by leakage of trace live virus. In the experimental animals, mice were subcutaneously injected with a higher dose of the vaccine, and the experimental animals survived healthy during a longer observation period. 2) The vaccine of the present invention is suitable for large-scale production by the method of genetic engineering, which reduces the cost. 3) Infected animals and immunized animals can be distinguished by using the vaccine of the present invention. In nature, animals infected with type A foot-and-mouth disease will produce type A foot-and-mouth disease antibodies in their bodies; animals infected with type O foot-and-mouth disease will produce type O foot-and-mouth disease antibodies in their bodies; animals infected with Asia type I foot-and-mouth disease will produce Asia I in their bodies FMD antibodies. Vaccine of the present invention has O type, A type and Asia I type three kinds of foot-and-mouth disease virus VP1 immune determinants simultaneously, after immunization injection, produce anti-O type, anti-A type, anti-Asia I type three kinds of foot-and-mouth disease antibodies in the animal body, can In this way, infected animals can be distinguished from immune animals, and confusion can be avoided in the import and export of animals. 4) The vaccine of the present invention contains three kinds of foot-and-mouth disease virus VP1 immunodeterminant of O type, A type and Asia I type, and contains the T-Helper polypeptide of VP4 again, greatly strengthens the effect of neutralizing antibody, and 4 repeat fragments are expressed, The molecular weight of the polypeptide is large, the half-life in the body is prolonged, and the time for producing neutralizing antibodies is also greatly extended.

Description of drawings

Fig. 1 is the structural SEQ ID in the expression vector of genetically engineered bacterium;

Fig. 2 is a schematic diagram of the construction process of plasmid F4, illustrating the process of how to concatenate gene fragments four times;

Fig. 3 is the growth pattern of the genetically engineered bacteria (E. Coli) containing the Lac vector during the fermentation process (12 hr, fermenter: Swiss Bio, 100 L).

Detailed ways

Embodiment one:

According to the amino acid sequence of the VP1 immune determinant of O, A and Asia I three types of FMDV and the T-cell helper of VP4, the present invention designs and synthesizes DNA gene fragments. One genetically engineered bacterium is a 15-amino acid fragment of the foot-and-mouth disease virus strain encoding the T-cell helper of VP4, which is linked to the DNA sequence of the immunogenic immunodeterminant region in the O-type VP1 protein and linked to the Asia I-type VP1 protein. The DNA sequence of the immunogenic immunodeterminant region is connected to the DNA sequence of the immunogenic immunodeterminant region in the A-type VP1 protein as a repeating fragment, and the nucleotide sequence of the repeating fragment is shown in FIG. 1 .

The novel trivalent foot-and-mouth disease genetically engineered polypeptide vaccine of the present invention contains 2n ^-1 polypeptides encoded by the nucleic acid sequence shown in SEQ ID in series, wherein n is an integer of 1-5. SEQ ID contains the DNA sequence of 15 amino acid fragments of the T-cell helper encoded by the foot-and-mouth disease virus strain of VP4, which is connected to the DNA sequence of the immunogenic immunodeterminant region in the O-type VP1 protein as a repeated fragment, which is connected in series 4 times, Its expression product can stimulate the production of antibodies against O-type, Asia I and A-type foot-and-mouth disease viruses.

Preferably, the vaccine of the present invention contains 2 ^n-1 polypeptides encoded by SEQ ID in series, wherein n is an integer of 2-4.

Most preferably, the vaccine of the present invention contains 2 ^n-1 polypeptides encoded by SEQ ID in series, wherein n is 3.

In order to improve the bioavailability and enhance the immune effect, the vaccine of the present invention may also contain pharmaceutically acceptable carriers, adjuvants and/or immune adjuvants and other substances.

An immune adjuvant is a substance that can enhance the immune response, which can be mixed with the antigen, facilitates the deposition or pooling of the injected substance, and can also enhance the antibody response.

Substances that can be used as immune adjuvants include: 1) Microorganisms and their products, such as mycobacteria, Corynebacterium pumilus, pertussis bacilli and lipopolysaccharides from the extracts of Zoran-negative bacilli, muramyl di peptide etc. 2) Polynucleotides, such as polyinosinic acid, polyadenylic acid, poly-L-glutamic acid, etc. 3) Freund's adjuvant (Freund's adjuvant), including incomplete Freund's adjuvant (made by mixing equal amounts of antigen aqueous solution and oil (paraffin oil or vegetable oil), and then emulsifier (lanolin or Tween 80) water-in-oil antigen emulsion) and complete Freund's adjuvant (adding mycobacteria such as BCG to incomplete adjuvant). 4) Inorganic substances, such as alum and aluminum hydroxide, etc.

In recent years, it has been found that the following substances can also be used as immune adjuvants, including: 1) Bacterial toxins, such as cholera toxin (CT) and Escherichia coli heat-labile toxin (LT). 2) Attenuated derivatives or variants of CT and LT. 3) Human endogenous immune regulatory factors, such as IL-2, IL-12, GM-GSF. 4) Hormones. 5) Lipopeptides. 6) Saponin, saponin derivative QS-21. 7) Synthetic oligonucleotide fragment (CpG ODN) containing CpG motif. 8) Derivatives of lipid A, such as lipopolysaccharide derivative monophosphoryl lipid A (MPL). 9) Derivatives of muramyl dipeptide (MDP).

In addition, certain delivery systems with intrinsic immunostimulatory activity can also be used as immune adjuvants for vaccine construction, these delivery systems include but not limited to: liposomes, emulsions, cochleates, virus particles, microparticles and Immunostimulatory complexes (ISCOMs).

All types of immune adjuvants mentioned above can be used in the present invention. The adjuvant can be used in combination with the immunogenic polypeptide of the present invention at an appropriate dose to form the vaccine of the present invention.

Preferably, the vaccine of the present invention contains incomplete Freund's adjuvant or aluminum hydroxide, more preferably, the vaccine of the present invention contains incomplete Freund's adjuvant as an immune enhancer. The ratio of lanolin and paraffin oil in incomplete Freund's adjuvant varies slightly with the seasons, for example, the ratio of lanolin and paraffin oil in spring, summer and autumn is about 3:7, and the ratio of the two in winter is about 1.5 : 8.5.

The preparation method of the genetic engineering vaccine of the present invention comprises the steps of directly synthesizing the nucleotide sequences shown in 2n ^-1 tandem SEQ IDs, or first synthesizing several DNA fragments, which are annealed and connected to produce the nucleotide sequences shown in the SEQ IDs. The DNA sequence shown. For the convenience of subsequent operations, when designing DNA fragments, suitable restriction sites can be introduced into both ends of the sequence, so that the target sequence can be cloned into a suitable vector.

In a preferred embodiment of the present invention, for the carrier of synthetic genetically engineered bacteria, the present invention designs and synthesizes the DNA sequence of 15 amino acid fragments of the T-cell helper of the foot-and-mouth disease virus strain encoding VP4 and connects the immunogen in the O-type VP1 protein The DNA sequence of the immunogenic immunodeterminant region in the Asian type I VP1 protein is connected to the DNA sequence of the immunogenic immunogenic determinant region in the type A VP1 protein as 1 repeat The fragments are produced by ligation of 4 DNA fragments after annealing, and each repeating fragment F1 contains the nucleic acid sequence shown in SEQ ID and a suitable restriction endonuclease cut point. F1 was cloned into the vector by genetic engineering, and then F2 (containing two serially connected F1 sequences) was cloned into the vector by restriction endonucleases with matching cohesive ends after digestion, and the expressed polypeptide contained 2 repeats (VP4+O-type VP1+Asia I-type VP1). According to a similar method, F4, F8, and F16 can be cloned into the vector in turn, which contain 4, 8, and 16 serial F1 sequences in sequence. After being expressed in a suitable expression system, the obtained polypeptides contain 4 , 8, and 16 tandem repeats (VP4+O-type VP1+Asia I-type VP1).

In the preparation method of the present invention, the expression system used for polypeptide expression may be a prokaryotic expression system or a eukaryotic expression system. The expression system includes a suitable host cell, and a plasmid or vector capable of replicating and stably existing in the host cell.

Examples of host cells include, but are not limited to: bacterial cells, such as Escherichia coli, Streptococcus, Salmonella typhimurium, etc.; eukaryotic cells, such as yeast, etc.

Vectors that may be used may include DNA sequences of chromosomal, non-chromosomal and synthetic origin. Such as: phage DNA, baculovirus, bacterial plasmid, yeast plasmid, and vectors derived from a combination of plasmid, phage, and viral DNA.

Preferably, the polypeptides of the invention are expressed in prokaryotic expression systems. More preferably, the polypeptide of the present invention can be cloned into a high-efficiency expression vector (such as a commercially available expression vector) for fusion expression with a carrier protein.

The present invention also relates to a genetically engineered bacterial strain, the plasmid carried by it contains 2 ^n-1 nucleic acid sequences shown in concatenated SEQ ID, wherein n is an integer of 1-5.

Preferably, the plasmid carried by the engineering bacteria contains 2 ^n-1 nucleic acid sequences shown in concatenated SEQ ID, wherein n is an integer of 2-4.

More preferably, the plasmid carried by the engineering bacteria contains 2n ^-1 nucleic acid sequences shown in concatenated SEQ ID, wherein n is 3.

The invention also provides the use of the trivalent genetic engineering polypeptide vaccine in preventing and treating foot-and-mouth disease. The vaccine of the present invention can be injected into animals to stimulate the animals to produce specific anti-O type, anti-A type and anti-Asia I type three kinds of FMD antibodies, so the vaccine of the present invention can be used to distinguish immunized animals from infected animals .

Table 1 is the test results of the antigen-antibody reaction. Antibodies are O-type and A-type FMD standard serum and O-type FMD bovine serum are provided by Animal Husbandry and Veterinary Research Institute of Shanghai Academy of Agricultural Sciences. Antigens are self-prepared trivalent genetic engineering Peptide vaccine.

Table 1: Antigen-antibody responses

Embodiment two:

1: Construction of expression plasmids

The genetically engineered bacterium is Escherichia coli containing the gene of the trivalent genetically engineered polypeptide vaccine.

In order to synthesize the vector of genetically engineered bacteria, we linked the DNA sequence of the 15 amino acid fragments of the foot-and-mouth disease virus strain that encodes the T-cell helper of VP4 to the DNA sequence of the immunogenic immunodeterminant region in the O-type VP1 protein. The DNA sequence of the immunogenic immunodeterminant region in the type I VP1 protein is connected with the DNA sequence of the immunogenic immunodeterminant region in the type A VP1 protein as a repeat fragment, which is produced by annealing and ligation of 4 DNA fragments , each repeating fragment F1 contains the nucleic acid sequence shown in Figure 1 and a suitable restriction endonuclease site. Utilize the method of genetic engineering to clone F1 into the Lac promoter expression plasmid, then, according to the method in Figure 2, F2, F4 can be cloned into the Lac promoter expression plasmid in turn, they contain 2, 4 F1 sequences in series ( VP4+O type VP1+Asia I type VP1+A type VP1).

2: Cloning of the F4 genes of two genetically engineered bacteria in the expression plasmid pkk223-3

After the plasmid pkk223-3 was cut with restriction endonuclease BamH I/Sal I, the gene F4 was cloned into the pkk223-3 plasmid according to conventional methods to obtain the expression plasmid.

3: Transform the expression plasmid into Escherichia coli, culture it at 37°C, and induce it with IPTG at a final concentration of 0.2-0.5mmol/L.

4: fermentation

250ml of seed culture solution (1000ml of seed culture solution contains 10g of peptone, 5g of yeast extract, 20ml of 0.02mol/L phosphate buffer, pH7.0) in a 1000ml Erlenmeyer flask, sterilized at 120°C for 20 minutes, cooled and then added 20% glucose solution 5ml. Add 1 ml of the bacterial strain preserved in glycerol at low temperature to the above solution, add Ampicillin to make the final concentration 50 μg/ml, and cultivate on a shaker at 37° C. for 12-14 hours (150 rpm) as seed bacteria for expanded cultivation.

1000ml of seed culture solution contains 20g of peptone, 10g of yeast extract, 20ml of 0.2mol/L phosphate buffer, pH 7.0, sterilized at 120°C for 20 minutes, cooled to 37°C and then added with Ampicillin to make it the final concentration 50 μg/ml. Add 20ml of seed culture solution, then add 5ml of 20% glucose solution, and 1mg each of trace elements CaCl2, NiNO3, CoCl3, MgSO4, FeCl3, and maintain the listed conditions for fermentation (fermenter: 100L; temperature: 37°C; stirring speed: 700rpm; ventilation: 80L/min; pH7.0-7.5). Take 1ml of fermentation broth at regular time intervals and put them in 2 plastic centrifuge tubes, centrifuge at 8000rpm for 10min, remove the supernatant, weigh the weight of the bacteria as wet weight (g/L). As shown in Figure 3, the bacterial concentration peaked after 8-12 hours of fermentation.

After fermentation, the cells were collected by centrifugation at 4000rpm for 30min.

The bacteria were suspended in a solution containing 1% sodium chloride, 1mmol/L EDTA, and 20mmol/L potassium phosphate pH7.0 at a ratio of 1000g/3L, and 1g of lysozyme was added to the suspension, and stirred at room temperature for 1 hour , to break the cells, the cell suspension was centrifuged at 10000rpm for 30min, and the supernatant was discarded.

Add the above precipitate to 8mol/L urea solution at a ratio of 250g/L. Stir and extract overnight, centrifuge at 20,000rpm for 30min, take the supernatant, and precipitate after renaturation, centrifuge at 10,000rpm for 30min, take the precipitate, separate and purify the fusion protein, freeze-dry to obtain the product, and make a homogenate with sterile water , forming a trivalent polypeptide vaccine.

5: Detection of FMDV vaccine

(1), Reagent

The standard serum antibodies to type O and type A foot-and-mouth disease were provided by the Institute of Animal Husbandry and Veterinary Medicine, Shanghai Academy of Agricultural Sciences.

(2), sample

After fermenting the genetically engineered strain containing pkk223-3/F8, collect the bacteria by centrifugation, break the cells, extract with 8M urea, collect the precipitate after centrifugation, add 1ml of sterile water to 1mg, and take 1ml of each supernatant. Add O-type and A-type foot-and-mouth disease standard sera to the precipitation solution, shake well, produce a precipitation reaction, and the solution is turbid; add O-type and A-type foot-and-mouth disease standard serum to the supernatant solution, shake well, there is no precipitation reaction, and the solution is clear.

Results: The expressed fusion protein produced antigen-antibody reactions with O-type and A-type FMD standard sera, but the supernatant did not contain fusion protein, so it did not react with O-type and A-type FMD standard sera.

6: Antigen-antibody response of FMDV vaccine

The immunogenicity of the vaccine of the present invention can be determined by using the protein in the solution, which can react with the antigen and antibody to produce a precipitate.

(1) Antigen: the test product is the protein obtained after expression and purification of the genetically engineered bacteria strain of the present invention.

(2) Antibodies: Standard serum antibodies to Type O and Type A foot-and-mouth disease were provided by the Institute of Animal Husbandry and Veterinary Medicine, Shanghai Academy of Agricultural Sciences.

(3), antigen-antibody reaction: in the trivalent foot-and-mouth disease genetic engineering polypeptide vaccine, add O-type foot-and-mouth disease antibody and A-type foot-and-mouth disease antibody respectively, according to the result of Table 1, show that trivalent foot-and-mouth disease genetic engineering polypeptide vaccine is compatible with O-type antibody and A-type antibody All had immunoprecipitation reactions.

7: Vaccine safety experiment (mice experiment)

Twenty male Kunming mice, weighing about 20 g each, were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences. Divided into two groups, one group is the trivalent foot-and-mouth disease genetically engineered polypeptide vaccine, and the other group is the control group.

In the treatment group, 1 mg of the purified antigenic protein of the trivalent foot-and-mouth disease genetically engineered polypeptide vaccine was suspended in 1 ml of sterile water, and in the control group, 1 ml of normal saline was injected. Mice were injected intraperitoneally and observed for 24 hours. All mice survived, proving that the trivalent foot-and-mouth disease genetically engineered polypeptide vaccine is a safe drug. Survival rates are shown in the table below.

Survival rate Trivalent Foot-and-Mouth Disease Genetic Engineering Peptide Vaccine 10/10 control group 10/10

Claims (20)

1. a kind of foot-and-mouth disease virus trivalent polypeptide vaccine is prepared by 1 genetic engineering bacterium, and foot-and-mouth disease virus coat protein has four kinds namely VP1, VP2, VP3, VP4, and this genetic engineering bacterium is the T- The DNA sequence of the 15-amino acid fragment of the cell helper is connected to the DNA sequence of the immunogenic immunodeterminant region of the O-type VP1 protein. The DNA sequence of the immunogenic immunodeterminant region of the Asia I type VP1 protein is connected to the A-type The DNA sequence of the immunogenic immunodeterminant region in the VP1 protein is used as a repeating fragment, and the foot-and-mouth disease virus trivalent polypeptide vaccine contains 2 ^n-1 proteins encoded by the nucleic acid sequence shown in the sequence of SEQ ID, wherein n is an integer of 1-5. 2. foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 1, wherein: 1 genetically engineered bacterium contains the protein encoded by the nucleotide sequence shown in the SEQ ID of 2 ^n-1 series, wherein n is 2-4 integer. 3. foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 2, wherein: 1 genetically engineered bacterium contains the protein encoded by the nucleotide sequence shown in the SEQ ID of 2 ^n-1 series, wherein n is 3, and its molecular weight It is 38928. 4. foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 3, genetically engineered bacterium contains the protein encoded by the nucleotide sequence shown in 4 concatenated SEQID. 5. foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 4, the gene that contains tandem in the genetically engineered bacterium expresses with the Lac plasmid that carries in escherichia coli. 6. The foot-and-mouth disease virus trivalent polypeptide vaccine according to any one of claims 1-5, wherein: said vaccine further comprises a pharmaceutically acceptable carrier, adjuvant and/or immune adjuvant. 7. The foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 6, wherein said immune adjuvant is Freund's incomplete adjuvant. 8. The foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 6, wherein said immune adjuvant is Al(OH)3. 9. the foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 6, wherein said immune adjuvant is gamma-polyglutamic acid poly-gamma-glutamic acid. 10. a kind of preparation method of foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 1, comprises the nucleotide sequence shown in the SEQ ID of 2n ^-1 concatenations of genetically engineered bacteria cloned into carrier and expressed in expression system The steps, wherein n is an integer of 1-5. 11. the preparation method of foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 10, comprises the nucleotide sequence shown in the SEQ ID of 2 ^n-1 concatenations of genetically engineered bacteria cloned into carrier and expressed in expression system Step, wherein n is an integer of 2-4. 12. the preparation method of foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 11, comprises the nucleotide sequence shown in the SEQ ID of 2n ^-1 series connection of genetically engineered bacterium and is cloned into carrier and expresses in expression system step, where n is 3. 13. The method for preparing the foot-and-mouth disease virus trivalent polypeptide vaccine according to any one of claims 10-12, wherein the nucleic acid sequence is expressed in a prokaryotic expression system. 14. The method for preparing the foot-and-mouth disease virus trivalent polypeptide vaccine according to any one of claims 10-12, wherein the nucleic acid sequence is expressed in a eukaryotic expression system. 15. A genetically engineered strain of the foot-and-mouth disease virus trivalent polypeptide vaccine according to claim 1, the plasmid carried by it contains 2n ^-1 nucleic acid sequences shown in concatenated SEQ ID, wherein n is an integer of 1-5. 16. two kinds of genetically engineered bacterial strains of foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 15, the plasmid that it carries contains respectively the nucleic acid shown in 2n ^-1 sequenced SEQ ID No.1 and SEQ ID No.2 sequence, where n is an integer of 2-4. 17. the genetically engineered strain of foot-and-mouth disease virus trivalent polypeptide vaccine as claimed in claim 16, the plasmid that it carries contains the nucleotide sequence shown in the SEQ ID of 2n ^-1 series, wherein n is 3. 18. the foot-and-mouth disease virus trivalent polypeptide vaccine of claim 1, its purposes is: be used for preventing and treating O type, A type and Asia I foot-and-mouth disease. 19. The foot-and-mouth disease virus trivalent polypeptide vaccine according to claim 1, which is used for distinguishing infected animals from immunized animals. 20. A preparation method of foot-and-mouth disease virus polypeptide vaccine of artiodactyla domestic animals, comprising gene recombination and preparation of recombinant fusion protein, characterized in that: (1) For the synthesis of genetically engineered bacteria, the DNA sequence of the 15 amino acid fragments encoding the T-cell helper of VP4 was synthesized by chemical synthesis, and the DNA sequence of the immunogenic immunodeterminant region in the O-type VP1 protein was linked to Asia The DNA sequence of the immunogenic immunodeterminant region in the type I VP1 protein is connected with the DNA sequence of the immunogenic immunodeterminant region in the A type VP1 protein as a repeated fragment, which is connected in series 4 times; (2) For synthetic genetically engineered bacteria, insert 4 repeating fragments into the Lac plasmid vector; (3) Transforming the above-mentioned recombinant plasmid into Escherichia coli for expression to obtain a bacterial strain; (4) Ferment the bacterial strain in nutrient-rich LB medium, the fermentation temperature is 37°C, and the fermentation time is 12-24 hours. Add ampicillin to the fermentation broth to make the final concentration 50 μg/ml, and IPTG, the concentration is 0.5 mM, centrifuged after fermentation to obtain bacterial cells, crushed cells, renaturation of inclusion bodies, separation and purification of fusion protein, freeze-dried to obtain the product, and homogenized with adjuvant to form a trivalent polypeptide vaccine.

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