CN1287858C - Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method - Google Patents

Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method Download PDF

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CN1287858C
CN1287858C CN 02137011 CN02137011A CN1287858C CN 1287858 C CN1287858 C CN 1287858C CN 02137011 CN02137011 CN 02137011 CN 02137011 A CN02137011 A CN 02137011A CN 1287858 C CN1287858 C CN 1287858C
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foot
mouth disease
vaccine
polypeptide
amino acid
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CN1408349A (en
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郑兆鑫
严维耀
李光金
陈维灶
赵凯
刘明秋
张青
易建中
徐泉兴
盛祖恬
郭杰炎
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Fudan University
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Fudan University
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Abstract

The present invention relates to the design and the construction of a foot-and-mouth disease genetic engineering polypeptide vaccine and a preparation method thereof. In the present invention, a livestock IgG constant region of a heavy chain or beta-galactosidase is used as carrier protein is connected with a foot-and-mouth disease virus antigen polypeptide to prepare the novel foot-and-mouth disease genetic engineering polypeptide vaccine. Genes of the livestock IgG constant region of a heavy chain are amplified with a PCR method, and antigenic amino acid peptide segment genes in encoded foot-and-mouth disease virus VP1 protein are synthetized by chemical synthesis, connected in series in a multicopy mode and connected with the genes of the IgG constant region of a heavy chain or beta-galactosidase genes to form a fusion gene. The fusion gene is inserted into an expression vector and fermented to obtain the vaccine. The vaccine has the advantages of high safety, obvious effects and ideal protective effects on livestock.

Description

A kind of foot-and-mouth disease gene engineering polypeptide vaccine and preparation method thereof
Technical field
The invention belongs to field of genetic engineering, being specifically related to a kind of is foot-and-mouth disease gene engineering polypeptide vaccine of carrier protein and preparation method thereof with susceptible animal autoimmune globulin IgG CH or beta galactosidase.
Background technology
World Organization for Animal Health classifies foot and mouth disease first of the category-A infectious disease as.Foot and mouth disease is cloven-hoofed animals such as livestock contagious disease the most serious in the world today, main harm pig, cattle, sheep.For many years, foot and mouth disease is large-scale outbreak and popular worldwide, causes the tremendous economic loss to animal husbandry.Prophylactic immunization is the main means of this virus of control.In the vaccine of various prevention foot and mouth disease, recombinant vaccine is easy to preserve because safety is good, and advantages such as effect stability have broad application prospects.
(foot-and-mouth disease virus, FMDV) immunogenicity is closely-related is its coat protein VP1 gene, contains foot and mouth disease virus major antigen epi-position in this albumen with foot and mouth disease virus.Comparatively sophisticated at present foot and mouth disease virus gene engineered vaccine is the reorganization polypeptide vaccine, is about to foot and mouth disease virus major antigen epi-position and links to each other with a macromolecular carrier albumen, constitutes a fusion rotein, and then brings out animal generation immunne response effectively.
Summary of the invention
The objective of the invention is to propose with from body IgG or beta galactosidase as safe and reliable foot and mouth disease polypeptide vaccine of carrier, preparation and preparation method thereof.
The foot and mouth disease polypeptide vaccine that the present invention proposes, be to be carrier protein with domestic animal autoimmune globulin IgG CH or beta galactosidase, the foot-and-mouth disease virus antigen polypeptide chain is connected on the appropriate location (as N end, C end or in the centre position) of carrier protein, constitutes fusion rotein.Wherein the foot-and-mouth disease virus antigen polypeptide adopts in the foot and mouth disease virus VP1 albumen and has antigenic amino acid peptide section, and it is formed the cascaded structure of multicopy, adds amino acid polypeptide in peptide section junction as joint.
Can be discerned and submission by the animal body immune system better in order to guarantee fusion rotein, carrier protein as this fusion rotein can be part section or the full length sequence of domestic animal from body IgG CH or beta galactosidase, and can be domestic animal IgG CH full length amino acid sequence the 30th to 328 of homologous sequence, IgG CH the homologous sequence of totally 299 amino acid peptide sections or beta galactosidase from the 1st to the 583rd the homologous sequence of totally 583 amino acid peptide sections.Domestic animal is from 329 aminoacid of body IgG CH total length, and the IgG CH nucleotide homology of different cloven-hoofed animal origins is greater than 85%, and amino acid identity is greater than 90%; And 1024 aminoacid of beta galactosidase total length, different colibacillary beta galactosidase nucleotide homologies are greater than 90%, and amino acid identity is greater than 95%.For the immunogenicity of the antigen polypeptide that guarantees to insert, can choose different antigen and insert the site, the foot-and-mouth disease virus antigen polypeptide that will have cascaded structure inserts wherein.And antigen polypeptide can be connected the appropriate location of carrier protein, inserts as N end, C end or in the centre position.
Among the present invention, antigen polypeptide adopts in the foot and mouth disease virus VP1 albumen has antigenic amino acid peptide section, adopts the multicopy cascaded structure simultaneously.For having antigenic amino acid peptide section, as O type foot and mouth disease virus VP1 protein 21~40,141~160,200~213 amino acids peptide sections, and can before and after the float amino acid residue of suitable number, as adopting amino acid peptide sections such as 35~53,134~158,141~158,135~144,188~209; Aminoacid sequence in the foot and mouth disease virus antigen epitope partly has conservative, and as the RGD site among 141~160AA, but not conservative part is a foundation with the epidemic isolates gene order; Usually as among 21~40AA have 1-4 aminoacid to be replaced, have 1-5 aminoacid to be replaced among 141~160AA, have 1-2 aminoacid to be replaced among 200~213AA, its principle is that these amino acid peptides Duan Jun has antigenicity.And for the cascaded structure of antigen polypeptide, (or the three copy cascaded structures of 200~213AA)-141~160AA also can adopt the cascaded structure of other multicopies (as two copies, four copies etc.) as 141~160AA-21~40AA; In the cascaded structure, having antigenic a certain amino acid peptide section can reuse, and residing position can be different.When having antigenic amino acid peptide section and being connected to form cascaded structure, add suitable amino acid polypeptide as joint in peptide section junction, aminoacid kind and number in the joint can change as required, and its principle is can show enough antigenicities after forming fusion rotein with the IgG CH.
Among the embodiment foot-and-mouth disease virus antigen polypeptide chain is connected on the C end of carrier protein, constitutes fusion rotein, its structural representation is as follows:
Figure C0213701100041
After making antigen polypeptide be expressed as fusion rotein, can embody antigenicity better, we have suitably chosen a certain section in IgG CH and the beta galactosidase as carrier protein, adopt respectively among the embodiment as described later the 30th to 328 of pig IgG CH totally 299 amino acid peptide sections and escherichia coli beta galactosidase from the 1st to the 583rd totally 583 amino acid peptide sections as carrier protein: the FMDV antigen polypeptide then adopts 3 major antigen epi-positions the O type foot and mouth disease virus VP1 albumen, promptly 21~40,141~160,200~213 3 amino acid peptide sections, and with its composition 141~160AA-21~40AA (or cascaded structure of 200~213AA)-141~160AA, add suitable amino acid polypeptide as joint in peptide section junction, adopt the antigen polypeptide structure of 141~160AA (" Y ")-Pro-Gly (" M ")-21~40AA (" X ")-Gln-Phe-Glu-Leu-Glu-Phe-Met-Val-Pro-Ser-Arg (" N ")-141~160AA (" Y ") as described later among the embodiment, former and later two joints are respectively 2 and 11 aminoacid.
The present invention in an embodiment, adopt the FMDV antigen gene sequences with cascaded structure to be: SEQ ID NO.1, the antigen polypeptide aminoacid sequence of its coding are SEQ ID NO.2; SEQ ID NO.3, the antigen polypeptide aminoacid sequence of its coding are SEQ ID NO.4; SEQ ID NO.5, the antigen polypeptide aminoacid sequence of its coding is SEQID NO.6.Its architectural feature is as follows:
Respectively there is the DNA sequence of coding 141~160 amino acids of a copy at the two ends of antigen gene, and the centre is 21~40 of the codings of a copy or the DNA sequence of 200~213 amino acids; Two joints that connect these sequences are respectively 2 and 11 aminoacid.
On gene level, SEQ ID NO.1 is connected the C-terminal of IgG CH, forms fusion gene, express obtaining fusion rotein.This fusion rotein has following characteristics:
(1) on gene level, carrier protein gene size is about 900bp, and antigenic peptides gene size is about 240bp, and whole fusion gene size is about 1.2kb.
(2) on protein level, carrier protein only comprises the 30th of pig IgG CH to the 328th amino acids peptide section, i.e. 299 aminoacid; Antigen polypeptide is above-mentioned tandem polypeptide section, and wherein 21~40 amino acids peptide sections are t cell epitopes, 141~160 amino acids peptide sections, one B cell epitope.Whole fusion protein molecule amount is about 50kDa.
On gene level, SEQ ID NO.3 and SEQ ID NO.5 are connected the C-terminal of beta galactosidase, form fusion gene, express obtaining fusion rotein.This fusion rotein has following characteristics:
(1) on gene level, carrier protein gene size is about 1.8kb, and antigenic peptides gene size is about 200-240bp, and whole fusion gene size is about 2kb.
(2) on protein level, carrier protein only comprise beta galactosidase from the 1st to the 583rd totally 583 amino acid peptide sections; Antigen protein is above-mentioned tandem polypeptide, and whole fusion protein molecule amount is about 72kDa.
The invention allows for the preparation method of above-mentioned foot-and-mouth disease gene engineering polypeptide vaccine, comprise gene preparation, reorganization and Expression of Fusion Protein etc., its step is as follows: from animal spleen, amplify the IgG weight chain constant area gene with the RT-PCR method, beta-galactosidase gene adopt in the expression vector from carries gene.DNA sequence with having antigenic amino acid peptide section in the chemical synthesis process composite coding VP1 albumen connects into complete antigen gene on dna level, this gene is linked to each other with above-mentioned carrier protein gene, constitutes fusion gene; Then above-mentioned fusion gene is inserted the expression plasmid carrier, and change coli strain over to.Bacterial strain is placed bacteria culture media, and cultivation temperature is between 30 ℃ to 37 ℃, and incubation time 8 to 25 hours is collected thalline then; With bacterial cell disruption, collect fusion rotein, fusion rotein is made into oil emulsion, can obtain novel foot and mouth disease polypeptide vaccine.
The new generation vaccine that the present invention proposes is used for immune susceptible animal, the generation of prevention foot and mouth disease.Consider to make the maximum immunological effect of vaccine performance and keep the longest effect persistency that we propose: the foot and mouth disease polypeptide vaccine that is carrier protein with certain animal IgG CH is best suited for this kind of prevention animal foot and mouth disease.As the IgG CH with pig is that the foot and mouth disease polypeptide vaccine of carrier protein is best suited for the prevention Schweineseuche; And be that the foot and mouth disease polypeptide vaccine of carrier protein is best suited for prevention cattle foot and mouth disease with the IgG CH of cattle.But also cross-application simultaneously is that the foot and mouth disease polypeptide vaccine of carrier protein also is applicable to his class zooprophylazis foot and mouth disease of immunity with a class animal IgG CH promptly.
This new generation vaccine is used to prevent dissimilar foot and mouth disease according to antigen gene (or polypeptide) the sequence difference that makes up.The same immunological effect of considering vaccine, we propose: with O type FMDV gene order serves as to be best suited for prevention O type foot and mouth disease according to the vaccine that makes up; With A type FMDV gene order serves as to be best suited for prevention A type foot and mouth disease according to the vaccine that makes up; In like manner, serve as to be best suited for according to the vaccine that makes up to prevent the foot and mouth disease of type separately with various FMDV gene orders such as AsiaI.
The another feature of this new generation vaccine is: have cross immunity power.Promptly the FMDV gene order with a certain strain in a certain type is the infection that is widely used in the different strains of prevention homotype foot and mouth disease virus in various susceptible animals according to the vaccine that makes up.
The vaccine that the present invention proposes has adopted technique for gene engineering and methods such as PCR, gene clone and sequencing, sequence analysis, gene recombinaton in preparation and checkout procedure.And use the SDS-PAGE method of protein detection, immunological methods such as Western blotting, T cell proliferation experiment, Cavia porcellus and pig, the detection of cattle anti-virus ability have detected the effect of this vaccine.
Among the embodiment (), be that carrier protein makes up Schweineseuche O-shaped polypeptide vaccine pXZ860 with IgG.Its result is as follows:
SDS-PAGE electrophoresis detection proof is expressed the fusion protein molecule amount and the consistent (see figure 1) of expection that obtains." 1 " is protein molecular weight marker among the figure; " 2,3,4 " are respectively the protein expression bands that blank acceptor bacterium Top10, blank plasmid pTrcHis transform Top10 and recombiant plasmid pXZ860 conversion Top10.The visible molecular weight fusion rotein consistent in band " 4 " with expection.
Western blotting detects this fusion rotein of proof and has very strong antigenicity, can special antigen antibody reaction (see figure 2) take place with standard antibody." 1,2,3,4 " are corresponding with " 1,2,3,4 " in " Fig. 1 " among Fig. 2.Visible special immunoblotting in the band " 4 " wherein.
With this vaccine immunity Cavia porcellus, detect the T cell proliferation.The result proves this vaccine induced animal T cell proliferation effectively, and then inducing cell immunity and humoral immunization (see figure 3).Abscissa among the figure is represented standard antigen extension rate X, and vertical coordinate is represented stimulation index SI, and (annotate: stimulation index is with the cpm value and the ratio value representation that does not add antigenic negative control group cpm value of experimental group; Cpm is the per minute umber of pulse of measuring with liquid scintillation counter).As seen 160 times of standard antigen dilutions just can be stung the Cavia porcellus T cell proliferation (SI>2 expression positive reactions) that the menstruating on time after pregnancy vaccine immunity is crossed.
With this vaccine immunity Cavia porcellus, attack with O type foot and mouth disease virus behind the certain hour.The result proves that this vaccine has good protection effect to Cavia porcellus, and protective rate reaches 100% (seeing Table 1).
With this vaccine immunity pig, attack with O type foot and mouth disease virus behind the certain hour.The result proves that equally this vaccine has good protection effect to pig, and protective rate reaches 100% (seeing Table 2).
Table 1 pXZ860 Seedling is to the immune protective effect of Cavia porcellus
Vaccine Vaccine immunity dosage Virus attack dosage The experimental guinea pig number Morbidity Cavia porcellus number Protective rate
The immune group matched group 400ug 0 50ID 50/0.1ml 50ID 50/0.1ml 6 6 0 6 100% 0
Table 2 pXZ860 Seedling is to the immune protective effect of pig
Vaccine Vaccine immunity dosage Virus attack dosage Attack the pig's head number Morbidity pig's head number Protective rate
The immune group matched group 7mg 0 100ID 50/2ml 100ID 50/2ml 5 5 0 5 100% 0
Among the embodiment (two), be that carrier protein makes up cattle foot and mouth disease O type polypeptide vaccine pXZ880 with the beta galactosidase.Its result is as follows:
SDS-PAGE electrophoresis detection proof is expressed the fusion protein molecule amount and the consistent (see figure 4) of expection that obtains." 1,1 ' " is protein molecular weight marker among the figure; " 2,3,4 " are respectively recombiant plasmid pXZ880 and blank plasmid pWR590 transformed into escherichia coli JM101, and the protein expression band of blank acceptor bacterium JM 101, the fusion rotein consistent with expection of visible molecular weight shown in the arrow in band " 2 "." 2 ' " is the occlusion body electrophoretic band; " 3 ' " is fusion rotein purification band, and the purified back of this fusion rotein purity is greater than 95%.
Western blotting detects this fusion rotein of proof and has very strong antigenicity, can special antigen antibody reaction (see figure 4) take place with standard antibody." 5,6,7 " are and " 2,3,4 " corresponding Western blotting reaction band among the figure; " 4 ', 5 ', 6 ' " is the Western blotting reaction band corresponding with " 3 ', 3,4 ".Wherein band " 5 " and visible special immunoblotting in " 4 ' ".
With this vaccine immunity Cavia porcellus, detect the T cell proliferation.The result proves this vaccine induced animal T cell proliferation effectively, and then inducing cell immunity and humoral immunization (see figure 5).Abscissa among the figure is represented standard antigen extension rate X, and vertical coordinate is represented stimulation index SI, and (annotate: stimulation index is with the cpm value and the ratio value representation that does not add antigenic negative control group cpm value of experimental group; Cpm is the per minute umber of pulse of measuring with liquid scintillation counter).As seen 160 times of standard antigen dilutions just can be stung the Cavia porcellus T cell proliferation (SI>2 expression positive reactions) that the menstruating on time after pregnancy vaccine immunity is crossed.
With the fusion rotein preparation vaccine of purification, and, attack with O type foot and mouth disease virus behind the certain hour with this vaccine immunity Cavia porcellus.The vaccine that result proof contains 100ug fusion rotein dosage just can reach 80% (seeing Table 3) to the protective rate of Cavia porcellus.
Directly prepare vaccine with occlusion body, and, attack with cattle source O type foot and mouth disease virus behind the certain hour with this vaccine immunity cattle of doses.The result proves that equally this vaccine has the better protect effect to cattle, and protective rate reaches 66.7% (seeing Table 4).
Table 3 pXZ880 Seedling is to the immanoprotection action of Cavia porcellus
Vaccine Vaccine immunity dosage Virus attack dosage Attack a Cavia porcellus number A morbidity Cavia porcellus number Protective rate
The immune group matched group 100ug 0 50ID 50/0.2ml 50ID 50/0.2ml 5 5 1 5 80% 0
Table 4 pXZ880 Seedling is to the immanoprotection action of cattle
Vaccine Vaccine immunity dosage Virus attack dosage Attack the ox head number Morbidity ox head number Protective rate
The immune group matched group 10mg 0 100ID 50/2ml 100ID 50/2ml 3 2 1 2 66.7% 0
Among the embodiment (three), be that carrier protein makes up Schweineseuche O-shaped polypeptide vaccine pXZ870 with the beta galactosidase.Its result is as follows:
SDS-PAGE electrophoresis detection proof is expressed the fusion protein molecule amount and the consistent (see figure 6) of expection that obtains." 1 " is protein molecular weight marker among the figure; " 2,3,4 " are respectively recombiant plasmid pXZ870 and blank plasmid pWR590 transformed into escherichia coli JM101, and the protein expression band of blank acceptor bacterium JM101, the fusion rotein consistent with expection of visible molecular weight shown in the arrow in band " 2 ".
Western blotting detects this fusion rotein of proof and has stronger antigenicity, can special antigen antibody reaction (see figure 6) take place with standard antibody." 5,6,7 " are and " 2,3,4 " corresponding Western blotting reaction band, wherein visible special immunoblotting in the band " 5 " among the figure.
Directly prepare vaccine with occlusion body, and, attack with pig source O type foot and mouth disease virus behind the certain hour with this vaccine immunity pig of doses.The result shows that this vaccine has good protection effect to pig, and protective rate reaches 100% (seeing Table 5).
Table 5 pXZ870 Seedling is to the immune protective effect of pig
Vaccine Vaccine immunity dosage Virus attack dosage Attack the pig's head number Morbidity pig's head number Protective rate
The immune group matched group 7mg 0 100ID 50/2ml 100ID 50/2ml 5 5 0 5 100% 0
Description of drawings
Fig. 1 is the SDS-PAGE electrophoresis diagram of polypeptide vaccine pXZ860.
Fig. 2 is that the Western blotting of polypeptide vaccine pXZ860 detects diagram.
Fig. 3 detects T cell diagram behind the polypeptide vaccine pXZ860 immune guinea pig.
Fig. 4 among Fig. 4 (a) is the SDS-PAGE electrophoresis detection diagram of polypeptide vaccine pXZ860, and Fig. 4 (b) is that the Western blotting of this vaccine detects diagram.
Fig. 5 detects T cell diagram behind the polypeptide vaccine pXZ860 immune guinea pig.
Fig. 6 is that SDS-PAGE electrophoresis and the Western blotting of polypeptide vaccine pX870 detects diagram.
The specific embodiment
Embodiment (one): with anti-Schweineseuche O-shaped polypeptide vaccine pXZ860 is that example specifically describes the present invention.
With the RT-PCR one-step method IgG weight chain constant area gene that from pig spleen, increases.
Encode in the chemosynthesis pig O type foot and mouth disease virus VP1 gene DNA sequence of 21~40,141~160 amino acids peptide sections and the DNA sequence of joint amino acid peptide section, series connection becomes the primary structure (SEQ ID NO.1) of 141~160AA-21~40AA-141~160AA.Plasmid vector pTrcHis with digestion with restriction enzyme, is mixed this fragment with the above-mentioned IgG weight chain constant area gene and the antigen gene of connecting, under effects such as T4 ligase, coupled reaction takes place.The recombinant DNA that obtains, the competent cell of transformed into escherichia coli Top10 bacterial strain is incubated in the ampicillin flat board, and 37 ℃ of inversions are spent the night.Arbitrarily the picking transformant is identified with enzyme action respectively, dna sequencing Analysis and Identification transformant, thus obtain positive colony.The gene order that the sequence analysis proof is inserted conforms to design.With this clone's called after pXZ860.
Is fermentation liquid with pXZ860 with the LB culture fluid that contains the 50ug/ml ampicillin, and 37 ℃ of mixing speed 6000rpm aerobic culture 2 hours add IPTG and induce after 10 hours and collect thalline.Again thalline is suspended in the shell-broken liquid Ultrasound Instrument breaking cellular wall of usefulness 95W power 5 minutes.Ultrasonic back 5000rpm collected inclusion body in centrifugal 20 minutes.And occlusion body is made into oil emulsion, can obtain a kind of novel Schweineseuche O-shaped gene engineering polypeptide vaccine.
The related fusion rotein SDS-PAGE electrophoresis detection of this vaccine the results are shown in shown in the accompanying drawing 1; Western blotting testing result is seen shown in the accompanying drawing 2; Detect the T cell proliferation experiment behind this vaccine immunity Cavia porcellus, the results are shown in shown in the accompanying drawing 3; The anti-virus ability testing result sees Table 1 and table 2.
Embodiment (two): with anti-cattle foot and mouth disease O type polypeptide vaccine pXZ880 is that example further describes the present invention.
With encode in the synthetic VP1 gene of chemical method segmental DNA sequence of 21~40,141~160 amino acids and linker DNA sequence, series connection becomes the primary structure (SEQ IDNO.3) of 141~160AA-21~40AA-141~160AA, is inserted into expression vector pWR590.Get 1ug pWR590 with EcoR I and Bamll I double digestion, in 37 ℃ of reactions two hours, big fragment was reclaimed in the electrophoresis rubber tapping.This large fragment DNA is mixed with the above-mentioned fragment of connecting, under effects such as T4 ligase, coupled reaction takes place.The recombinant DNA transformed into escherichia coli competent cell that obtains is incubated in the ampicillin flat board, and 37 ℃ of inversions are spent the night.Picking white transformant with BstEII single endonuclease digestion, EcoR I and Bamll I double digestion, pcr amplification, dna sequencing Analysis and Identification transformant, thereby obtains positive colony.The gene order that the sequence analysis proof is inserted conforms to implementation sequence.With this clone's called after pXZ880.
Is fermentation liquid with pXZ880 with the LB culture fluid that contains the 50ug/ml ampicillin, and 37 ℃ of mixing speed 6000rpm aerobic culture 2 hours add IPTG and induce after 10 hours and collect thalline.Again thalline is suspended in the shell-broken liquid Ultrasound Instrument breaking cellular wall of usefulness 95W power 5 minutes.Ultrasonic back 5000rpm collected inclusion body in centrifugal 20 minutes.With the inclusion body cracking, carry out purification through the Sepharose chromatographic column.At last the fusion rotein of occlusion body or purification is made into oil emulsion and can obtains novel cattle foot and mouth disease O type gene engineering polypeptide vaccine.
Embodiment (three): with anti-Schweineseuche O-shaped polypeptide vaccine pXZ870 is that example further describes the present invention.
With encode in the synthetic VP1 gene of chemical method segmental DNA sequence of 200~213,141~160 amino acids and linker DNA sequence, series connection becomes the primary structure (SEQID NO.5) of 141~160AA-200~213AA-141~160AA, is inserted into expression vector pWR590.Make up and screening positive clone with the similar method of embodiment (two).The gene order that the sequence analysis proof is inserted conforms to implementation sequence.With this clone's called after pXZ870.
Is fermentation liquid with pXZ870 with the LB culture fluid that contains the 50ug/ml ampicillin, and 37 ℃ of mixing speed 6000rpm aerobic culture 2 hours add IPTG and induce after 10 hours and collect thalline.Again thalline is suspended in the shell-broken liquid Ultrasound Instrument breaking cellular wall of usefulness 95W power 5 minutes.Ultrasonic back 5000rpm collected inclusion body in centrifugal 20 minutes.Inclusion body is made into oil emulsion, can obtains a kind of novel Schweineseuche O-shaped gene engineering polypeptide vaccine.
The sequence that the present invention relates to is 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:
VSNVRGDLQVLAQKAERVLPPGETQVQRRQHTDISFILDRFVQFELEFMVPSRVSNVRGDLQVLAQKAERVLP
SEQ ID NO.3:
GTT ACC AAC GTT CGT GGT GAC CTG CAG GTT CTG GCT CAG AAA GCT GCT
CAA TGG TTG CAA GCA CCA CTG GAC GTC CAA GAC CGA GTC TTT CGA CGA
CGT ACC CTG CCG CCG GGT GAA ACC CAG GTT CAG CGT CGT CAG CAC ACC
GCA TGG GAC GGC GGC CCA CTT TGG GTC CAA GTC GCA GCA GTC GTG TGG
GAC GTT TCT TTC ATC CTG GAC CGT TTC GTT CAG TTC GAA CTG GAG TTC
CTG CAA AGA AAG TAG GAC CTG GCA AAG CAA GTC AAG CTT GAC CTC AAG
ATG GTT CCG TCT CGT GTT ACC AAC GTT CGT GGT GAC CTG CAG GTT CTG
TAC CAA GGC AGA GCA CAA TGG TTG CAA GCA CCA CTG GAC GTC CAA GAC
GCT CAG AAA GCT GCT CGT ACC CTG CCG
CGA GTC TTT CGA CGA GCA TGG TAC GGC
SEQ ID NO.4:
VTNVRGDLQVLAQKAARTLPPGETQVQRRQHTDVSFILDRFVQFELEFMVPSRVTNVRGDLQVLAQKAARTLP
SEQ ID NO.5:
GTA TCT AAC AAA CGT GGT GAC CTG CAG GTA CTT GCT CAG AAA GCT GAA CGT GCT
CAT AGA TTG TTT GCA CCA CTG GAC GTC CAT GAA CGA GTC TTT CGA CTT GCA CGA
CTG CCG CCC GGT CGT CAC AAA CAG AAA ATC GTA GCT CCG GCT AAA CAG CTG CTG
GAC GGC GGG CCA GCA GTG TTT GTC TTT TAG CAT CGA GGC CGA TTT GTC GAC GAC
CAA TTC GAG CTG GAA TTC ATG GTA CCC TCT CGT GTA TCT AAC AAA CGT GGT GAC
GTT AAG CTC GAC CTT AAG TAC CAT GGG AGA GCA CAT AGA TTG TTT GCA CCA CTG
CTG CAG GTA CTT GCT CAG AAA GCT GAA CGT GCT CTG CCG
GAC GTC CAT GAA CGA GTC TTT CGA CTT GCA CGA GAC GGC
SEQ ID NO.6:
VSNKRGDLQVLAQKAERALPPGRHKQKIVAPAKQLLQFELEFMVPSRVSNKRGDLQVLAQKAERALP

Claims (4)

1, a kind of foot-and-mouth disease gene engineering polypeptide vaccine, it is characterized in that be carrier protein with domestic animal from body IgG, the foot-and-mouth disease virus antigen polypeptide chain is connected on the C-terminal of carrier protein, constitute a fusion rotein, wherein antigen polypeptide adopts in the foot and mouth disease virus VP1 albumen and has antigenic amino acid peptide section, and it is formed the cascaded structure of multicopy, add amino acid polypeptide in peptide section junction as joint.
2, polypeptide vaccine according to claim 1, the carrier protein that it is characterized in that fusion rotein are 29 aminoacid full length sequences of domestic animal IgG CH3, or the 30th to 328 totally 299 amino acid peptide sections.
3, polypeptide vaccine according to claim 1, it is characterized in that antigen polypeptide adopts following cascaded structure: Y-M-X-N-Y or Y-M-Z-N-Y, wherein, " X " of same cascaded structure, " Y ", " Z " peptide section derives from same type foot and mouth disease virus VP1 protein sequence, and " X ", " Y ", " Z " peptide section is represented foot and mouth disease O type virus VP 1 protein 21 へ 40 respectively, 141 へ 160,200 へ, 213 amino acids peptide sections, perhaps foot and mouth disease A, the corresponding amino acid peptide section of AsiaI type virus surface proteins, " M " is " Pro-Gly " two peptide linkers, and " N " is " Gln-Phe-Glu-Leu-Glu-Phe-Met-Val-Pro-Ser-Arg " 11 peptide linkers.
4, polypeptide vaccine according to claim 1 is characterized in that described to have antigenic peptide section be SEQ IDNO.2, or SEQ ID NO.4, or SEQ ID NO.6.
CN 02137011 2002-09-16 2002-09-16 Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method Expired - Fee Related CN1287858C (en)

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CN100381170C (en) * 2003-09-03 2008-04-16 上海华谊生物技术有限公司 Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use
CN1318592C (en) * 2003-11-11 2007-05-30 中国农业科学院兰州兽医研究所 O type foot and mouth disease virus DNA vaccine and its preparing method
CN100342911C (en) * 2004-01-13 2007-10-17 厦门大学 Bivalent DNA vaccine of type A and type O foot-and-mouth disease virus and its preparing process
CN101659696B (en) * 2008-08-27 2013-03-13 中牧实业股份有限公司 Asia I-type aftosa synthetic peptide vaccine
CN101659695B (en) * 2008-08-27 2012-08-29 中牧实业股份有限公司 O-type aftosa synthetic peptide vaccine
CN101721698B (en) 2008-10-24 2014-04-02 法罗斯疫苗公司 Foot-and-mouth disease resistant vaccine composition and preparation and application thereof
CN101643500B (en) * 2009-05-19 2012-06-06 中牧实业股份有限公司 Asia I synthetic peptide vaccine of foot and mouth disease
CN118304393A (en) * 2024-01-19 2024-07-09 中国农业科学院兰州兽医研究所(中国动物卫生与流行病学中心兰州分中心) O-type foot-and-mouth disease virus composite epitope protein vaccine for current epidemic strains and application

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