CN1408349A - Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method - Google Patents

Abstract

The invention relates to the design, construction and preparation method of a foot-and-mouth disease genetic engineering polypeptide vaccine. The invention adopts livestock IgG heavy chain constant region or beta-galactosidase as carrier protein, connects it with foot-and-mouth disease virus antigen polypeptide, and prepares novel foot-and-mouth disease genetic engineering polypeptide vaccine. The IgG heavy chain constant region gene of livestock was amplified by PCR method, and the antigenic amino acid peptide gene encoding the VP1 protein of foot-and-mouth disease virus was synthesized by chemical synthesis, and these genes were connected in multiple copies, and then combined with the IgG heavy chain The constant region gene or the β-galactosidase gene are connected to form a fusion gene. The fusion gene is inserted into an expression vector and fermented to prepare a vaccine. The vaccine has good safety, remarkable effect and ideal protective effect on livestock.

Description

A kind of foot-and-mouth disease genetic engineering polypeptide vaccine and preparation method thereof

technical field

The invention belongs to the field of genetic engineering, and specifically relates to a foot-and-mouth disease genetically engineered polypeptide vaccine with susceptible animal autoimmune globulin IgG heavy chain constant region or beta-galactosidase as carrier protein and a preparation method thereof.

Background technique

The International Veterinary Bureau ranks foot-and-mouth disease as the first class A infectious disease. Foot-and-mouth disease is the most serious livestock infectious disease in the world today, mainly harming pigs, cattle, sheep and other cloven-hoofed animals. For many years, foot-and-mouth disease has broken out and become popular in a large scale all over the world, causing huge economic losses to animal husbandry. Vaccination is the main means of controlling the virus. Among various vaccines for preventing foot-and-mouth disease, genetically engineered vaccines have broad application prospects due to their advantages of good safety, easy preservation and stable effect.

Closely related to the immunogenicity of foot-and-mouth disease virus (FMDV) is its coat protein VP1 gene, which contains the main epitope of foot-and-mouth disease virus. At present, the relatively mature foot-and-mouth disease virus genetic engineering vaccine is a recombinant polypeptide vaccine, which links the main antigenic epitope of foot-and-mouth disease virus with a large molecule of carrier protein to form a fusion protein, which can effectively induce immune response in animals.

Contents of the invention

The object of the invention is to propose a safe and reliable foot-and-mouth disease polypeptide vaccine and a preparation method thereof using autologous IgG or beta-galactosidase as a carrier.

The foot-and-mouth disease polypeptide vaccine that the present invention proposes is to take domestic animal autologous immunoglobulin IgG heavy chain constant region or β-galactosidase as carrier protein, and foot-and-mouth disease virus antigen polypeptide is connected in the appropriate position of carrier protein (such as N terminal, C terminal or in the middle position), constituting the fusion protein. The foot-and-mouth disease virus antigen polypeptide adopts the antigenic amino acid peptide segment in the foot-and-mouth disease virus VP1 protein, and forms a multi-copy serial structure, and adds amino acid peptides as linkers at the peptide segment connections.

In order to ensure that the fusion protein can be better recognized and presented by the immune system of the animal body, the carrier protein of the fusion protein can be a partial or full-length sequence of the constant region of the heavy chain of the IgG heavy chain of the livestock itself or β-galactosidase, And it can be the homologous sequence of the full-length amino acid sequence of the constant region of the IgG heavy chain of livestock, the homologous sequence of a total of 299 amino acid peptides from the 30th to the 328th of the IgG heavy chain constant region, or the β-galactosidase from the The homologous sequence of 583 amino acid peptides from position 1 to position 583. The full length of the IgG heavy chain constant region of domestic animals is 329 amino acids. The nucleotide homology of the IgG heavy chain constant region from different artiodactyls is greater than 85%, and the amino acid homology is greater than 90%. The length is 1024 amino acids, the nucleotide homology of β-galactosidase of different Escherichia coli is greater than 90%, and the amino acid homology is greater than 95%. In order to ensure the immunogenicity of the inserted antigen polypeptide, different antigen insertion sites can be selected, and the foot-and-mouth disease virus antigen polypeptide with a tandem structure is inserted therein. The antigenic polypeptide can be connected to the appropriate position of the carrier protein, such as N-terminal, C-terminal or inserted in the middle.

In the present invention, the antigenic polypeptide adopts the antigenic amino acid peptide segment in the foot-and-mouth disease virus VP1 protein, and simultaneously adopts a multi-copy serial structure. For amino acid peptides with antigenicity, such as 21ヘ40, 141ヘ160, 200ヘ213 amino acid peptides of O-type foot-and-mouth disease virus VP1 protein, and an appropriate number of amino acid residues can be floated back and forth, such as 35ヘ53 , 134ヘ1 58, 141ヘ158, 135ヘ144, 188ヘ209 and other amino acid peptides; the amino acid sequence part of the FMD virus epitope is conservative, such as the RGD site in 141ヘ160AA, not conservative Partly based on the gene sequence of popular strains; usually, 1-4 amino acids can be replaced in 21ヘ40AA, 1-5 amino acids can be replaced in 141ヘ160AA, and 1-2 amino acids can be replaced in 200ヘ213AA Can be replaced, the principle is that these amino acid peptides are all antigenic. For the tandem structure of the antigenic polypeptide, such as the three-copy tandem structure of 141ヘ160AA-21ヘ40AA (or 200ヘ213AA)-141ヘ160AA, other multi-copy (such as two-copy, four-copy, etc.) tandem structures can also be used ; In the tandem structure, a certain amino acid peptide segment with antigenicity can be used repeatedly, and the positions can be different. When the antigenic amino acid peptides are connected to form a tandem structure, an appropriate amino acid polypeptide is added as a linker at the link of the peptides. The type and number of amino acids in the linker can be changed as needed. The principle is to form a fusion protein with the IgG heavy chain constant region After that, it can show sufficient antigenicity.

In the embodiment, the foot-and-mouth disease virus antigen polypeptide is connected to the C-terminus of the carrier protein to form a fusion protein, and its structure is shown as follows:

In order to better reflect the antigenicity after expressing the antigenic polypeptide into a fusion protein, we appropriately selected a certain segment of the constant region of the IgG heavy chain and β-galactosidase as the carrier protein, as described in the examples below. A total of 299 amino acid peptides from the 30th to 328th positions of the porcine IgG heavy chain constant region and a total of 583 amino acid peptides from the 1st to the 583rd positions of Escherichia coli β-galactosidase were used as carrier proteins; the FMDV antigen polypeptide was Three main antigenic epitopes in the VP1 protein of type O foot-and-mouth disease virus, namely 21ヘ40, 141ヘ160, and 200ヘ213 three amino acid peptides, were used to form 141ヘ160AA-21ヘ40AA (or 200ヘ213AA) The tandem structure of -141ヘ160AA, adding appropriate amino acid polypeptides as linkers at the connection of peptide segments, as in the following examples using 141ヘ160AA("Y")-Pro-Gly("M")-21ヘ40AA(" X")-Gln-Phe-Glu-Leu-Glu-Phe-Met-Val-Pro-Ser-Arg ("N")-141ヘ160AA ("Y") antigen polypeptide structure, the two linkers before and after are respectively 2 and 11 amino acids.

In the embodiment of the present invention, the sequence of the FMDV antigen gene with a tandem structure is: SEQ ID NO.1, the amino acid sequence of the antigen polypeptide encoded by it is SEQ ID NO.2; SEQ ID NO.3, the amino acid sequence of the antigen polypeptide encoded by it The sequence is SEQ ID NO.4; SEQ ID NO.5, and the amino acid sequence of the encoded antigen polypeptide is SEQ ID NO.6. Its structural characteristics are as follows:

At both ends of the antigen gene, there is a copy of the DNA sequence encoding 141ヘ160 amino acids, and in the middle is a copy of the DNA sequence encoding 21ヘ40 or 200ヘ213 amino acids; the two linkers connecting these sequences are 2 and 11 amino acids.

At the gene level, SEQ ID NO.1 is connected to the C-terminal of the IgG heavy chain constant region to form a fusion gene, and the fusion protein is obtained by expression. The fusion protein has the following characteristics: (1) At the gene level, the size of the carrier protein gene is about 900bp, the size of the antigen peptide gene is about 240bp, and the size of the entire fusion gene is about 1.2kb. (2) At the protein level, the carrier protein only contains the 30th to 328th amino acid peptide of the porcine IgG heavy chain constant region, that is, 299 amino acids; the antigenic polypeptide is the above-mentioned tandem peptide, of which 21ヘ40 amino acid peptide The segment is a T cell epitope, and the 141ヘ160 amino acid peptide segment is a B cell epitope. The molecular weight of the whole fusion protein is about 50kDa.

At the gene level, connect SEQ ID NO.3 and SEQ ID NO.5 to the C-terminus of β-galactosidase to form a fusion gene, and express to obtain a fusion protein. The fusion protein has the following characteristics: (1) At the gene level, the size of the carrier protein gene is about 1.8kb, the size of the antigen peptide gene is about 200-240bp, and the size of the entire fusion gene is about 2kb. (2) At the protein level, the carrier protein only contains 583 amino acid peptides from position 1 to position 583 of β-galactosidase; the antigenic protein is the above-mentioned tandem polypeptide, and the molecular weight of the entire fusion protein is about 72kDa.

The present invention also proposes a preparation method of the above-mentioned foot-and-mouth disease genetically engineered polypeptide vaccine, including gene preparation, recombination and fusion protein expression, etc., the steps are as follows: amplify the IgG heavy chain constant region gene from the animal spleen by RT-PCR method, The β-galactosidase gene adopts its own gene in the expression vector. Synthesize the DNA sequence encoding the antigenic amino acid peptide segment in the VP1 protein by chemical synthesis, link it into a complete antigen gene at the DNA level, link this gene with the above-mentioned carrier protein gene to form a fusion gene; then insert the above-mentioned fusion gene Expression plasmid vectors were transformed into E. coli strains. Place the bacterial strain in a bacterial culture medium, cultivate the temperature between 30°C and 37°C, and cultivate for 8 to 25 hours, then collect the bacteria; break the bacteria, collect the fusion protein, and formulate the fusion protein into an oil emulsion, namely A new type of foot-and-mouth disease peptide vaccine is available.

The novel vaccine proposed by the invention is used to immunize susceptible animals and prevent the occurrence of foot-and-mouth disease. In consideration of maximizing the immune effect of the vaccine and maintaining the longest duration of effect, we propose that the FMD polypeptide vaccine using the IgG heavy chain constant region of a certain animal as the carrier protein is most suitable for preventing FMD in this animal. For example, the foot-and-mouth disease polypeptide vaccine with the IgG heavy chain constant region of pigs as the carrier protein is most suitable for preventing porcine foot-and-mouth disease; while the foot-and-mouth disease polypeptide vaccine with the IgG heavy chain constant region of cattle as the carrier protein is most suitable for preventing bovine foot-and-mouth disease. At the same time, it can also be cross-applied, that is, the foot-and-mouth disease polypeptide vaccine using the IgG heavy chain constant region of one type of animal as the carrier protein is also suitable for immunizing other types of animals to prevent foot-and-mouth disease.

The novel vaccine is used to prevent different types of foot-and-mouth disease according to the sequence of the constructed antigen gene (or polypeptide). Also considering the immune effect of the vaccine, we propose that the vaccine constructed based on the O-type FMDV gene sequence is most suitable for preventing type O foot-and-mouth disease; the vaccine constructed based on the A-type FMDV gene sequence is most suitable for preventing type A foot-and-mouth disease; similarly , the vaccines constructed on the basis of the gene sequences of various types of FMDV such as AsiaI are most suitable for preventing respective types of foot-and-mouth disease.

Another feature of the novel vaccine is that it has cross-immunity. That is, the vaccine constructed on the basis of the FMDV gene sequence of a certain strain in a certain type is widely applicable to preventing the infection of different strains of the same type of foot-and-mouth disease virus in various susceptible animals.

During the preparation and inspection process of the vaccine proposed by the present invention, genetic engineering techniques and methods such as PCR, gene cloning and sequence determination, sequence analysis, and gene recombination are used. The efficacy of the vaccine was tested by immunological methods such as SDS-PAGE protein detection method, Western blotting, T cell proliferation experiment, guinea pig, pig and cattle anti-virus ability detection.

In the embodiment (1), the porcine foot-and-mouth disease O-type polypeptide vaccine pXZ860 was constructed with IgG as the carrier protein. The result is as follows:

SDS-PAGE electrophoresis detection proved that the molecular weight of the fusion protein obtained by expression was consistent with expectations (see Figure 1). "1" in the figure is the protein molecular weight marker; "2, 3, 4" are the protein expression bands of the blank recipient strain Top10, the blank plasmid pTrcHis transformed Top10 and the recombinant plasmid pXZ860 transformed Top10, respectively. A fusion protein with the expected molecular weight is seen in lane "4".

Western blotting test proves that the fusion protein has strong antigenicity and can react with the standard antibody for specific antigen-antibody reaction (see Figure 2). "1, 2, 3, 4" in Figure 2 corresponds to "1, 2, 3, 4" in "Figure 1". Specific immunoblotting can be seen in band "4".

Guinea pigs were immunized with the vaccine, and T cell proliferation was detected. The results proved that the vaccine can effectively induce the proliferation of animal T cells, and then induce cellular immunity and humoral immunity (see Figure 3). The abscissa in the figure represents the standard antigen dilution factor X, and the ordinate represents the stimulation index SI (note: the stimulation index is represented by the ratio of the cpm value of the experimental group to the cpm value of the negative control group without antigen; Measured pulses per minute). It can be seen that diluting the standard antigen 160 times can stimulate the proliferation of T cells in guinea pigs immunized by the vaccine (SI > 2 indicates a positive reaction).

Guinea pigs were immunized with the vaccine and challenged with type O foot-and-mouth disease virus after a certain period of time. The results prove that the vaccine has a good protective effect on guinea pigs, and the protection rate reaches 100% (see Table 1).

Pigs are immunized with this vaccine and challenged with O-type foot-and-mouth disease virus after a certain period of time. The results also proved that the vaccine has a good protective effect on pigs, and the protection rate reaches 100% (see Table 2).

Table 1 The immune protection effect of pXZ860 vaccine on guinea pigs Vaccine immunization dose Virus challenge dose Experimental guinea pig number Diseased guinea pig number Protection rate Immune group 400ug 50ID ₅₀ /0.1ml 6 0 100% control group 0 50ID ₅₀ /0.1ml 6 6 0

Table 2 The immune protection effect of pXZ860 vaccine on pigs Vaccine immunization dose Virus challenge dose Number of challenged pigs Number of diseased pigs Protection rate Immune group 7mg 100ID ₅₀ /2ml 5 0 100% control group 0 100ID ₅₀ /2ml 5 5 0

In Example (2), the bovine foot-and-mouth disease type 0 polypeptide vaccine pXZ880 was constructed with β-galactosidase as the carrier protein. The result is as follows:

SDS-PAGE electrophoresis detection proved that the molecular weight of the fusion protein obtained by expression was consistent with expectations (see Figure 4). "1, 1'" in the figure are protein molecular weight markers; "2, 3, 4" are the protein expression bands of Escherichia coli JM101 transformed with recombinant plasmid pXZ880 and blank plasmid pWR590, and the blank recipient strain JM 101, respectively. The fusion protein with the expected molecular weight can be seen indicated by the arrow in "2". "2'" is the inclusion body electrophoresis band; "3'" is the fusion protein purification band, and the purity of the fusion protein after purification is greater than 95%.

Western blotting test proves that the fusion protein has strong antigenicity and can react with the standard antibody specifically (see Figure 4). "5, 6, 7" in the figure are the Western blotting reaction bands corresponding to "2, 3, 4"; "4', 5', 6'" are the Western blotting reaction bands corresponding to "3', 3, 4" blotting reaction bands. Specific western blots can be seen in bands "5" and "4'".

Guinea pigs were immunized with the vaccine, and T cell proliferation was detected. The results proved that the vaccine can effectively induce the proliferation of animal T cells, and then induce cellular immunity and humoral immunity (see Figure 5). The abscissa in the figure represents the standard antigen dilution factor X, and the ordinate represents the stimulation index SI (note: the stimulation index is represented by the ratio of the cpm value of the experimental group to the cpm value of the negative control group without antigen; Measured pulses per minute). It can be seen that diluting the standard antigen 160 times can stimulate the proliferation of T cells in guinea pigs immunized by the vaccine (SI > 2 indicates a positive reaction).

The purified fusion protein was used to prepare a vaccine, and the vaccine was used to immunize guinea pigs, and after a certain period of time, the guinea pigs were challenged with O-type foot-and-mouth disease virus. The result proves that the vaccine containing 100ug of fusion protein dose has a protection rate of 80% for guinea pigs (see Table 3).

The vaccine is prepared directly with the inclusion body, and a certain dose of the vaccine is used to immunize cattle, and after a certain period of time, it is challenged with the cattle-derived O-type foot-and-mouth disease virus. The results also prove that the vaccine has a good protective effect on cattle, with a protection rate of 66.7% (see Table 4).

Table 3 The immune protection effect of pXZ880 vaccine on guinea pigs Vaccine immunization dose Virus challenge dose Number of guinea pigs attacked Number of guinea pigs with disease Protection rate Immune group 100ug 50ID ₅₀ /0.2ml 5 1 80% control group 0 50ID ₅₀ /0.2ml 5 5 0

Table 4 The immune protection effect of pXZ880 vaccine on cattle Vaccine immunization dose Virus challenge dose Number of challenged cows Number of sick cows Protection rate Immune group 10mg 100ID ₅₀ /2ml 3 1 66.7% Control group 0 100ID ₅₀ /2ml 2 2 0

In Example (3), the porcine foot-and-mouth disease O-type polypeptide vaccine pXZ870 was constructed with β-galactosidase as the carrier protein. The result is as follows:

SDS-PAGE electrophoresis detection proved that the molecular weight of the fusion protein obtained by expression was consistent with expectations (see Figure 6). "1" in the figure is the protein molecular weight marker; "2, 3, 4" are the protein expression bands of the recombinant plasmid pXZ870 and the blank plasmid pWR590 transformed into Escherichia coli JM101, and the blank recipient strain JM 101, respectively. In the band "2" The fusion protein with the expected molecular weight can be seen as indicated by the middle arrow.

Western blotting test proves that the fusion protein has strong antigenicity and can have specific antigen-antibody reaction with the standard antibody (see Figure 6). "5, 6, 7" in the figure are the Western blotting reaction bands corresponding to "2, 3, 4", and specific immunoblotting can be seen in band "5".

Vaccines are prepared directly with inclusion bodies, pigs are immunized with a certain dose of the vaccine, and after a certain period of time, they are challenged with porcine-origin O-type foot-and-mouth disease virus. The results showed that the vaccine had a good protective effect on pigs, with a protection rate of 100% (see Table 5).

Table 5 The immune protection effect of pXZ870 vaccine on pigs Vaccine immunization dose Virus challenge dose Number of challenged pigs Number of diseased pigs Protection rate Immune group 7mg 100ID ₅₀ /2ml 5 0 100% control group 0 100ID ₅₀ /2ml 5 5 0

Description of drawings

Fig. 1 is a schematic diagram of SDS-PAGE electrophoresis of the polypeptide vaccine pXZ860.

Figure 2 is a schematic diagram of Western blotting detection of the polypeptide vaccine pXZ860.

Fig. 3 is a diagram showing the detection of T cells after immunization of guinea pigs with polypeptide vaccine pXZ860.

Fig. 4(a) in Fig. 4 is an SDS-PAGE electrophoresis detection diagram of the polypeptide vaccine pXZ860, and Fig. 4(b) is a Western blotting detection diagram of the vaccine.

Fig. 5 is a diagram showing the detection of T cells after immunization of guinea pigs with polypeptide vaccine pXZ860.

Figure 6 is a schematic diagram of SDS-PAGE electrophoresis and Western blotting detection of the polypeptide vaccine pX870.

Detailed ways

Embodiment (1): The present invention is specifically described by taking the anti-foot-and-mouth disease O-type polypeptide vaccine pXZ860 as an example.

IgG heavy chain constant region gene was amplified from pig spleen by RT-PCR in one step.

Chemically synthesize the DNA sequence encoding the 21ヘ40, 141ヘ160 amino acid peptides and the DNA sequence of the linker amino acid peptides in the VP1 gene of porcine O-type foot-and-mouth disease virus, and concatenate them into the first level of 141ヘ160AA-21ヘ40AA-141ヘ160AA Structure (SEQ ID NO.1). Digest the plasmid vector pTrcHis with restriction endonucleases, mix the fragment with the above-mentioned IgG heavy chain constant region gene and tandem antigen gene, and perform ligation reaction under the action of T4 ligase and the like. The obtained recombinant DNA was transformed into competent cells of the Escherichia coli Top10 strain, cultured on an ampicillin plate, and inverted overnight at 37°C. Randomly pick transformants, identify them by restriction enzyme digestion, and identify transformants by DNA sequencing analysis, so as to obtain positive clones. Sequence analysis proved that the inserted gene sequence was consistent with the design. This clone was named pXZ860.

The pXZ860 was fermented with LB broth containing 50ug/ml ampicillin, cultured at 37°C with agitation speed of 6000rpm for 2 hours, and induced by adding IPTG for 10 hours to collect the bacteria. Then the bacteria were suspended in the wall-breaking solution, and the wall was broken for 5 minutes with a 95W power ultrasonic instrument. After sonication, the inclusion bodies were collected by centrifugation at 5000 rpm for 20 minutes. And formulating the inclusion body into oil emulsion can obtain a novel O-type genetically engineered polypeptide vaccine for porcine foot-and-mouth disease.

The fusion protein SDS-PAGE electrophoresis detection result involved in the vaccine is shown in Figure 1; the Western blotting test result is shown in Figure 2; the T cell proliferation experiment is detected after the vaccine is immunized with guinea pigs, and the results are shown in Figure 3; The test results of anti-virus ability are shown in Table 1 and Table 2.

Embodiment (2): The present invention is further described by taking the anti-bovine foot-and-mouth disease O-type polypeptide vaccine pXZ880 as an example.

The DNA sequence encoding the 21ヘ40 and 141ヘ160 amino acid fragments in the VP1 gene and the linker DNA sequence were synthesized by chemical methods, and connected in series to form the primary structure of 141ヘ160AA-21ヘ40AA-141ヘ160AA (SEQ ID NO.3), Insert it into the expression vector pWR590. Take 1 ug of pWR590 and digest it with EcoR I and BamH I, react at 37°C for two hours, and recover large fragments by electrophoresis and slicing. This large fragment of DNA is mixed with the above-mentioned tandem fragments, and a ligation reaction occurs under the action of T4 ligase and the like. The obtained recombinant DNA was transformed into Escherichia coli competent cells, cultured on ampicillin plates, and inverted overnight at 37°C. Pick the white transformants, and identify the transformants by single digestion with BstEII, double digestion with EcoR I and BamH I, PCR amplification, and DNA sequencing analysis to obtain positive clones. Sequence analysis proved that the inserted gene sequence was consistent with the designed sequence. This clone was named pXZ880.

The pXZ880 was cultured with LB culture medium containing 50ug/ml ampicillin as the fermentation medium at 37°C with agitation speed of 6000rpm for 2 hours, and was induced by adding IPTG for 10 hours to collect the bacteria. Then the bacteria were suspended in the wall-breaking solution, and the wall was broken for 5 minutes with a 95W power ultrasonic instrument. After sonication, the inclusion bodies were collected by centrifugation at 5000 rpm for 20 minutes. The inclusion bodies were lysed and purified by Sepharose chromatography. Finally, the inclusion body or the purified fusion protein is formulated into an oil emulsion to obtain a novel bovine foot-and-mouth disease O-type genetically engineered polypeptide vaccine.

Embodiment (3): Taking the anti-foot-and-mouth disease O-type polypeptide vaccine pXZ870 as an example to further describe the present invention.

The DNA sequence encoding the 200ヘ213, 141ヘ160 amino acid fragments in the VP1 gene and the linker DNA sequence were synthesized by chemical methods, and connected in series to form a primary structure of 141ヘ160AA-200ヘ213AA-141ヘ160AA (SEQID NO.5), Insert it into the expression vector pWR590. Positive clones were constructed and screened in a similar manner to Example (2). Sequence analysis proved that the inserted gene sequence was consistent with the designed sequence. This clone was named pXZ870.

The pXZ870 was fermented with LB broth containing 50ug/ml ampicillin, cultured with aeration at 37°C at a stirring speed of 6000rpm for 2 hours, and induced by adding IPTG for 10 hours, and the cells were collected. Then the bacteria were suspended in the wall-breaking solution, and the wall was broken for 5 minutes with a 95W power ultrasonic instrument. After sonication, the inclusion bodies were collected by centrifugation at 5000 rpm for 20 minutes. A novel O-type genetically engineered polypeptide vaccine for porcine foot-and-mouth disease can be obtained by formulating the inclusion body into an oil emulsion.

The sequences involved in the present invention are as follows:

SEQ ID NO.1:

GTG AGC AAC GTG AGG GGT GAC CTT CAA GTG TTG GCT CAG AAG GCA GAA AGA GTT

CAC TCG TTG CAC TCC CCA CTG GAA GTT CAC AAC CGA GTC TTC CGT CTT TCT CAA

CTG CCC CCC GGT GAG ACA CAG GTC CAG AGA CGC CAG CAC ACG GAT ATC TCG TTT

GAC GGG GGG CCA CTC TGT GTC CAG GTC TCT GCG GTC GTG TGC CTA TAG AGC AAA

ATA CTA GAC AGA TTT GTG CAG TTT GAG CTG GAG TTT ATG GTG CCC AGC AGG GTG

TAT GAT CTG TCT AAA CAC GTC AAA CTC GAC CTC AAA TAC CAC GGG TCG TCC CAC

AGC AAC GTG AGG GGT GAC CTT CAA GTG TTG GCT CAG AAG GCA GAA AGA GTT CTG

TCG TTG CAC TCC CCA CTG GAA GTT CAC AAC CGA GTC TTC CGT CTT TCT CAA GAC

CCC

GGG

SEQ ID NO.2:

VSNVRGDLQVLAQKAERVLPPGETQVQRRQHTDISFILDRFVQFELEFMVPSRVSNVRGDLQVLAQKAERVLPSEQ ID NO.3：GTT ACC AAC GTT CGT GGT GAC CTG CAG GTT CTG GCT CAG AAA GCT GCTCAA TGG TTG CAA GCA CCA CTG GAC GTC CAA GAC CGA GTC TTT CGA CGACGT ACC CTG CCG CCG GGT GAA ACC CAG GTT CAG CGT CGT CAG CAC ACCGCA TGG GAC GGC GGC CCA CTT TGG GTC CAA GTC GCA GCA GTC GTG TGGGAC GTT TCT TTC ATC CTG GAC CGT TTC GTT CAG TTC GAA CTG GAG TTCCTG CAA AGA AAG TAG GAC CTG GCA AAG CAA GTC AAG CTT GAC CTC AAGATG GTT CCG TCT CGT GTT ACC AAC GTT CGT GGT GAC CTG CAG GTT CTGTAC CAA GGC AGA GCA CAA TGG TTG CAA GCA CCA CTG GAC GTC CAA GACGCT CAG AAA GCT GCT CGT ACC CTG CCGCGA GTC TTT CGA CGA GCA TGG TAC GGCSEQ ID NO.4：VTNVRGDLQVLAQKAARTLPPGETQVQRRQHTDVSFILDRFVQFELEFMVPSRVTNVRGDLQVLAQKAARTLPSEQ ID NO.5: GTA TCT AAC AAA CGT GGT GAC CTG CAG GTA CTT GCT CAG AAA GCT GAA CGT GCTCAT AGA TTG TTT GCA CCA CTG GAC GTC CAT GAA CGA GTC TTT CGA CTT GCA CGACTG CCG CCC AT AA CT GGT ACGT C CCG GCT AAA CAG CTG CTGGAC GGC GGG CCA GCA GTG TTT GTC TTT TAG CAT CGA GGC CGA TTT GTC GAC GACCAA TTC GAG CTG GAA TTC ATG GTA CCC TCT CGT GTA TCT AAC AAA CGT GGT GACGTT AAG CTC GAC CTT AAG TAC CAT GGG AGA GCA CAT AGA TTG TTT GCA CCA CTGCTG CAG GTA CTT GCT CAG AAA GCT GAA CGT GCT CTG CCGGAC GTC CAT GAA CGA GTC TTT CGA CTT GCA CGA GAC GGCSEQ ID NO.6：VSNKRGDLQVLAQKAERALPPGRHKQKIVAPAKQLLQFELEFMVPSRVSNKRGDLQVLAQKAERALP

Claims (12)

1. The novel foot-and-mouth disease genetically engineered polypeptide vaccine is characterized in that the autologous IgG of domestic animals or the β-galactosidase of bacteria are used as the carrier protein, and the foot-and-mouth disease virus antigen polypeptide is connected to the end of the carrier protein to form a fusion protein, wherein the antigen polypeptide adopts foot-and-mouth disease There are antigenic amino acid peptides in the virus VP1 protein, and they are formed into a multi-copy tandem structure, and amino acid polypeptides are added as linkers at the junctions of the peptides. 2. The polypeptide vaccine according to claim 1, characterized in that the carrier protein of the fusion protein is the full-length sequence of 329 amino acids of the heavy chain constant region of livestock IgG, or a total of 299 amino acid peptides from position 30 to position 328. 3. The polypeptide vaccine according to claim 1, characterized in that the carrier protein of the fusion protein is a peptide segment of 583 amino acids from the 1st to the 583rd position of β-galactosidase. 4. The polypeptide vaccine according to claims 2 and 3, characterized in that the carrier protein of the fusion protein can be the homologous sequence of the full-length amino acid sequence of the constant region of the heavy chain of livestock IgG, the 30th to 328th position of the constant region of the IgG heavy chain A homologous sequence of a total of 299 amino acid peptides, or a homologous sequence of a total of 583 amino acid peptides from position 1 to position 583 of β-galactosidase. 5. The polypeptide vaccine according to claim 1, characterized in that the antigen polypeptide adopts the following tandem structure: Y-M-X-N-Y or Y-M-Z-N-Y, wherein the "X", "Y" and "Z" peptides of the same tandem structure are derived from the same type Foot-and-mouth disease virus VP1 protein sequence, and "X", "Y", and "Z" peptides represent the 21ヘ40, 141ヘ160, 200ヘ213 amino acid peptides of foot-and-mouth disease type O virus VP1 protein, or foot-and-mouth disease A, AsiaI Corresponding amino acid peptides of viral surface proteins, "M" is "Pro-Gly" dipeptide linker, "N" is "Gln-Phe-Glu-Leu-Glu-Phe-Met-Val-Pro-Ser-Arg" ten A peptide linker. 6. The polypeptide vaccine according to claim 5, characterized in that the "X", "Y", and "Z" peptide segments can float an appropriate number of amino acid residues: 35ヘ53, 134ヘ158, 141ヘ158, 135ヘ144, 188ヘ209 amino acid peptides. 7. The polypeptide vaccine according to claim 5 or 6, characterized in that the amino acids in the "X", "Y" and "Z" peptide segments can be adjusted according to the needs of preventing different mutant strains of foot-and-mouth disease: "X" 1-4 amino acids in the peptide can be substituted, 1-5 amino acids in the "Y" peptide can be substituted, and 1-2 amino acids in the "Z" peptide can be substituted. 8. The polypeptide vaccine according to any one of claims 1-3, characterized in that the antigenic polypeptide is linked to the C-terminus of the carrier protein. 9. The polypeptide vaccine according to claim 1, characterized in that the antigen polypeptide gene sequence is: SEQ ID NO.1, the amino acid sequence of the encoded antigen polypeptide is SEQ ID NO.2, SEQ ID NO.3, the encoded antigen The amino acid sequence of the polypeptide is SEQ ID NO.4, SEQ ID NO.5, and the amino acid sequence of the encoded antigen polypeptide is SEQ ID NO.6. 10. The application of the polypeptide vaccine as claimed in claim 1, characterized in that the foot-and-mouth disease virus polypeptide vaccine using the IgG heavy chain constant region of a certain susceptible animal as the carrier protein is suitable for preventing the foot-and-mouth disease of this animal; it can also be cross-applied: The foot-and-mouth disease virus polypeptide vaccine using the IgG heavy chain constant region of one type of susceptible animal as a carrier protein is also suitable for immunizing other types of susceptible animals to prevent foot-and-mouth disease. 11. The application of the polypeptide vaccine as claimed in claim 1, characterized in that the vaccine constructed on the basis of a certain type of foot-and-mouth disease virus gene sequence is most suitable for preventing this type of foot-and-mouth disease; and has cross-protection: a certain type of a certain The vaccine constructed on the basis of the foot-and-mouth disease virus gene sequence of the strain is widely applicable to various susceptible animals to prevent the infection of different strains of the same type of foot-and-mouth disease virus. 12. The preparation method of the foot-and-mouth disease genetically engineered polypeptide vaccine as claimed in claim 1, comprising gene preparation, recombination and expression of fusion protein, characterized in that the specific steps are as follows: (1) IgG heavy chain constant region gene was amplified from animal spleen by RT-PCR method. The DNA sequence encoding the antigenic amino acid peptide segment of the VP1 protein is synthesized by chemical synthesis, connected to a complete antigen gene at the DNA level, and this gene is connected to the above-mentioned IgG heavy chain constant region gene or β-galactosidase gene, constitute a fusion gene; (2) inserting the above-mentioned fusion gene into an expression plasmid vector, and transforming it into an E. coli strain; (3) Cultivate the positive strain at 30°C to 37°C for 8 to 25 hours, and then collect the bacteria; (4) The fusion protein expressed is collected by crushing the bacterium; (5) Purify the fusion protein and emulsify it to prepare the polypeptide vaccine related to the present invention.

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