CN1763105A - Fusion protein for stimulating organism to produce FMDV antibody and its coding gene and uses - Google Patents
Fusion protein for stimulating organism to produce FMDV antibody and its coding gene and uses Download PDFInfo
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- CN1763105A CN1763105A CN 200510108339 CN200510108339A CN1763105A CN 1763105 A CN1763105 A CN 1763105A CN 200510108339 CN200510108339 CN 200510108339 CN 200510108339 A CN200510108339 A CN 200510108339A CN 1763105 A CN1763105 A CN 1763105A
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Abstract
The present invention discloses one kind of fusion protein to excite organism to generate FMDV antibody and its coding gene and application. The fusion protein consists of VP1 protein of foot-and-mouth disease virus and at least one cell factor of GM-CSF and IFN-gamma. It has one of the following amino acid residue sequences: 1. SEQ ID No. 1 in the sequence list; and 2. SEQ ID No. 2 in the sequence list. The fusion protein of the present invention and its coding gene can excite animal body to generate FMDV antibody, has relative high immune protection against FMDV, and may be used in preventing and treating FMD. The fusion protein is stable, easy to preparation and suitable for industrial production. The present invention has important application foreground and practical significance in animal medicine field.
Description
Technical field
The present invention relates to fusion rotein and encoding gene thereof and application, particularly relate to a kind of fusion rotein and encoding gene and application of stimulating organism to produce FMDV antibody.
Background technology
(Foot and mouth disease FMD) is a kind of acute, hot and strong contagious disease of infecting both domestic animals and human to foot and mouth disease.This disease is popular in artiodactylous animals such as pig, ox, sheep, often causes huge loss to livestock industry.
Foot and mouth disease virus (FMDV) belongs to Picornaviridae (Picornaviridae), Hostis (Aphthovirus).It is human up to now find minimum animal virus, the about 22nm of diameter, molecular weight 8.08 * 10
6KDa.This virus contains 30%RNA, 70% protein.Single-stranded RNA (ss-RNA) is wire, be normal chain infect C-type virus C (+RNA), the about 2.6-2.8 of nucleic acid molecular weight * 10
6, based composition is A: G: U: C=26: 24: 22: 28.Mainly comprise VP
1, VP
2, VP
3, VP
4Four kinds of structural protein.Wherein, VP
1, VP
2, VP
3Form capsomere.VP
4Combining closely with RNA, is the virus particle internal component.
VP1 is one of main function antigen of foot and mouth disease virus, and granular leucocyte-macrophage colony stimulating factor (GM-CSF), interferon-(IFN-γ), interleukin cytokines such as (IL) are good molecular immune adjuvants.Foot-and-mouth disease gene engineering vaccine in the past is based upon on a kind of antigenic basis more, and immune protective effect is often undesirable.
Summary of the invention
The fusion rotein and the encoding gene thereof that the purpose of this invention is to provide a kind of stimulating organism to produce FMDV antibody.
Fusion rotein provided by the present invention is made up of at least a cytokine among the VP1 albumen of foot and mouth disease virus and GM-CSF, the IFN-γ.Described cytokine is preferably GM-CSF.
The VP1 albumen that can be foot and mouth disease virus in this fusion rotein is in aminoterminal, and cytokine is in carboxyl terminal, also can be that cytokine is in aminoterminal, and the VP1 albumen of foot and mouth disease virus is in carboxyl terminal.
Described fusion rotein is the protein with one of following amino acid residue sequences:
1) the SEQ ID NO:1 in the sequence table;
2) the SEQ ID NO:2 in the sequence table;
3) with the amino acid residue sequence of SEQ ID NO:1 or SEQ ID NO:2 through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of stimulating organism to produce FMDV antibody effect.
The protein that the amino acid residue sequence of SEQ ID NO:1 is made up of 359 amino-acid residues in the sequence table, from aminoterminal 1-143 position is the proteic amino acid residue sequence of cytokine GM-CSF, the amino acid residue sequence that is from the VP1 position of aminoterminal 149-359 foot and mouth disease virus, from aminoterminal 144-148 position is the amino acid residue sequence of connection peptides, 5 glycine of encoding will have the fusion rotein called after GM-CSF/VP1 of the amino acid residue sequence of SEQ ID NO:1 in the sequence table; The protein that the amino acid residue sequence of SEQ ID NO.2 is made up of 382 amino-acid residues in the sequence table, from aminoterminal 1-166 position is the amino acid residue sequence of cytokine IFN-γ, from aminoterminal 172-382 position is the proteic amino acid residue sequence of VP1 of foot and mouth disease virus, from aminoterminal 167-171 position is the amino acid residue sequence of connection peptides, 5 glycine of encoding will have the fusion rotein called after IFN-γ/VP1 of the amino acid residue sequence of SEQ ID NO:2 in the sequence table.
The gene of encoding said fusion protein is one of following dna sequence dna:
3) the SEQ ID NO:3 in the sequence table;
4) the SEQ ID NO:4 in the sequence table;
4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO:3 in the sequence table or SEQ ID NO:4.
The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.
The dna sequence dna of SEQ ID NO:3 is by 1077 based compositions in the sequence table, and its open reading frame is from 5 ' end the 1st to the 1077th bit base; From the sequence of 5 ' end the 1st to the 429th bit base for coding GM-CSF, 143 amino acid of encoding; From 5 ' end the 445th to the 1077th bit base is the proteic encoding sequence of VP1 of foot and mouth disease virus, 211 amino acid of encoding; From 5 ' end the 430th to the 444th bit base is the encoding sequence of connection peptides, 5 amino acid of encoding.
The dna sequence dna of SEQ ID NO:4 is by 1146 based compositions in the sequence table, and its open reading frame is from 5 ' end the 1st to the 1146th bit base; From the 1st to the 498th at 5 ' end, base is the encoding sequence of cytokine IFN-γ, 166 amino acid of encoding; From the VP1 proteic sequence of 5 ' end the 514th to the 1146th bit base for the coding foot and mouth disease virus, 211 amino acid of encoding; From the 499th to the 513rd at 5 ' end, base is the encoding sequence of connection peptides, 5 amino acid of encoding.
Contain the expression vector of the fusion rotein encoding gene of described stimulating organism to produce FMDV antibody, transgenic cell line and engineering bacteria also belong to protection scope of the present invention.
Another object of the present invention provides a kind of method of expressing the fusion rotein of above-mentioned stimulating organism to produce FMDV antibody.
Expression of Fusion Protein method provided by the present invention, be to make up the expression vector that contains described fusion rotein encoding gene, again the expression vector that makes up is imported host cell and obtain recombinant bacterial strain, cultivate this recombinant bacterial strain, from culture, isolate fusion rotein at last.
The fusion rotein of stimulating organism to produce FMDV antibody of the present invention and encoding gene thereof can be used for preventing and/or treating property of preparation aftosa vaccine.
The present invention forms as at least a cytokine among antigen and GM-CSF, the IFN-γ albumen VP1 of foot and mouth disease virus as immunological adjuvant fusion rotein and encoding gene thereof; can excite animal body to produce PMDV antibody; FMDV is had immune protective effect preferably, can be used in the prevention and treatment of FMD.This fusion rotein is stable, the preparation method is simple and easy to do, can be applicable to large-scale industrial production.The present invention has important application prospects and practical significance in the animal medicine field.
Below in conjunction with specific embodiment the present invention is further described.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The acquisition of the fusion rotein GM-CSF/VP1 of embodiment 1, the VP1 albumen that contains foot and mouth disease virus and GM-CSF
One, the construction of recombinant plasmid that contains the GM-CSF/VP1 antigen-4 fusion protein gene
1, the clone of VP1 and GM-CSF gene
1) clone of VP1 gene
Extract total RNA of FMDV, reverse transcription is cDNA and is template with it, under the guiding of upstream primer: 5 '-ACCACCTCTGCGGGTGAGTCT-3 ' and downstream primer: 5 '-CAGAAGCTGTTTTGCGGGT-3 ', increase with conventional PCR method, the PCR reaction conditions is: earlier 95 ℃ 5 minutes, 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute, 5 circulations; Again 94 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 1 minute, 28 circulations; Last 72 ℃ 10 minutes.After reaction finishes, the purpose fragment cloning of the VP1 gene of 633bp is gone among the carrier pGEM-TEasy (purchasing in company of Beijing fresh warp thread section), obtain containing the cloning vector of VP1 gene, called after pGEM-T Easy/VP1.
2) clone of GM-CSF gene
Extract the anticoagulant total RNA of ox, reverse transcription is cDNA and is template with it, under the guiding of upstream primer: 5 '-TCCTGCCAGCCCAAACATGAG-3 ' and downstream primer: 5 '-CTGGGCTTCAGACTTCAGATGTTAG-3 ', increase with conventional PCR method, the PCR reaction conditions is: earlier 95 ℃ 5 minutes, 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute, 5 circulations; Again 94 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 1 minute, 28 circulations; Last 72 ℃ 10 minutes.After reaction finishes, the purpose fragment cloning of the GM-CSF gene of 429bp is gone among the carrier pGEM-T Easy, obtain containing the cloning vector of VP1 gene, called after pGEM-T Easy/GM-CSF.
2, contain the acquisition of the recombinant plasmid of GM-CSF/VP1 antigen-4 fusion protein gene
1) the cloning vector pGEM-T Easy/VP1 that contains the VP1 gene that makes up with step 1 is a template, under the guiding of primer 5 '-GCAGAATTCACCACCTCTGCGGGTGAGTCT-3 ' and primer 5 '-GACCTCGAGCAGAAGCTGTTTTGCGGGT-3 ', with ExTaq PCR test kit (purchasing) subclone VP1 gene in company of Beijing fresh warp thread section; The cloning vector pGEM-T Easy/GM-CSF that contains the GM-CSF gene that makes up with step 1 is a template, under the guiding of primer 5 '-CTAGAATTCGGTGGCGGTGGCGGTGCACCTACTCGCCCACCCAA-3 ' and primer 5 '-TTACTCGAGCTTCTGGGCTGGTTCCCAG-3 ', with ExTaq PCR test kit subclone GM-CSF gene.The pcr amplification condition is: earlier 98 ℃ 5 minutes, 94 ℃ 1 minute, 62 ℃ 1 minute, 72 ℃ 1 minute, 5 circulations; Again 94 ℃ 1 minute, 57 ℃ 1 minute, 72 ℃ 1 minute, 28 circulations; Last 72 ℃ 10 minutes.
2) the VP1 gene being carried out enzyme with restriction enzyme BamH I and EcoR I cuts, the GM-CSF gene is carried out enzyme with restriction enzyme BamH I and EcoR I to be cut, after using restriction enzyme BamH I and EcoR I that carrier pGEX-6p-I (purchasing the company in Phamacia) enzyme is cut again, three kinds of enzymes are cut product T
4Dna ligase connects, and the ligation time is 16 hours.
3) with step 2) connection product transformed into escherichia coli DH5 α competent cell, single bacterium colony that picking grows shakes bacterium, the upgrading grain, carrying out double digestion with restriction enzyme BamHI and EcoRI identifies, cut through enzyme, obtain the positive cloned plasmids of 633bp and 429bp endonuclease bamhi, again this positive colony plasmid is done further evaluation with the method for PCR, the used primer sequence of PCR is 5 '-GCAGAATTCACCACCTCTGCGGGTGAGTCT-3 ' and 5 '-TTACTCGAGCTTCTGGGCTGGTTCCCAG-3 ', can amplify the segmental positive cloned plasmids of 1077bp, again it is carried out sequencing analysis, obtain the correct recombinant plasmid that contains the GM-CSF/VP1 antigen-4 fusion protein gene, called after pGEX-GM-CSF/VP1.
3, the coexpression of recombinant plasmid
The recombinant plasmid pGEX-GM-CSF/VP1 CaCl that contains the VP1/GM-CSF antigen-4 fusion protein gene with step 2 structure
2Method transformed into escherichia coli JM109 competent cell, the picking transformant, under 37 ℃ of conditions, induce coexpression albumen, inductor is IPTG, and using dosage is 100mg/L, induce end after, with urea renaturation inclusion body method expressed proteins is carried out purifying, obtaining molecular weight is the fusion rotein GM-CSF/VP1 of 65KD.
The plague protection experiment of embodiment 2, pGEX-GM-CSF/VP1 and GM-CSF/VP1
Recombinant plasmid pGEX-GM-CSF/VP1 and fusion rotein GM-CSF/VP1 difference immunized mice with embodiment 1 acquisition, with non-immunized mice is contrast, immunizing dose is 500 μ g/, every two all immunity once, immunity is 3 times altogether, and in each immunity back 2 week blood sampling separation of serum, measure serum antibody titer with the ELISA method, with foot and mouth disease inactivated vaccine (purchasing the company of in Beijing, herding) wrapper sheet, two anti-mark sheep anti-mouse antibody (purchasing in company of Beijing fresh warp thread section) for enzyme, and measurement result shows that the antibody titer of GM-CSF/VP1 immune serum can reach 1: 200; Block test kit (purchasing in Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences) with neutralizing antibody again and measure NAT, measurement result shows that GM-CSF/VP1 can stimulate the high-caliber neutralizing antibody of generation, and antibody titers can reach 1: 48; 2 all separating spleen lymphocytes carry out the T cell proliferation experiment with mtt assay after the last immunity, and the result shows that the T cell proliferative response significantly strengthens, and stimulation index is 0.348; Finish first three sky and carry out the delayed type hypersensitivity experiment, the result shows that the antigen-specific delayed type hypersensitivity is strong, and sole swelling degree is obvious.Experimental group detected result and control group are compared; the result is significantly improved by the mice serum IgG antibody horizontal of recombinant plasmid pGEX-GM-CSF/VP1 vaccine group that contains the VP1/GM-CSF antigen-4 fusion protein gene and coexpression Protein G M-CSF/VP1 vaccine group thereof; the T cell proliferative response significantly strengthens; antigen-specific delayed super quick (DHT) is strong; Th1 and Th2 cytokines expression amount have rising in various degree; the neutralizing antibody level significantly increases; show that its immune protective effect significantly improves with behind pGEX-GM-CSF/VP1 and the GM-CSF/VP1 immunized mice.
Sequence table
<160>4
<210>1
<211>359
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Met?Trp?Leu?Gln?Asn?Leu?Leu?Leu?Leu?Gly?Thr?Val?Val?Cys?Ser?Phe
1 5 10 15
Ser?Ala?Pro?Thr?Arg?Pro?Pro?Asn?Thr?Ala?Thr?Arg?Pro?Trp?Gln?His
20 25 30
Val?Asp?Ala?Ile?Lys?Glu?Ala?Leu?Ser?Leu?Leu?Asn?His?Ser?Ser?Asp
35 40 45
Thr?Asp?Ala?Val?Met?Asn?Asp?Thr?Glu?Val?Val?Ser?Glu?Lys?Phe?Asp
50 55 60
Ser?Gln?Glu?Pro?Thr?Cys?Leu?Gln?Thr?Arg?Leu?Lys?Leu?Tyr?Lys?Asn
65 70 75 80
Gly?Leu?Gln?Gly?Ser?Leu?Thr?Ser?Leu?Met?Gly?Ser?Leu?Thr?Met?Met
85 90 95
Ala?Thr?His?Tyr?Glu?Lys?His?Cys?Pro?Pro?Thr?Pro?Glu?Thr?Ser?Cys
100 105 110
Gly?Thr?Gln?Phe?Ile?Ser?Phe?Lys?Asn?Phe?Lys?Glu?Asp?Leu?Lys?Glu
115 120 125
Phe?Leu?Phe?Ile?Ile?Pro?Phe?Asp?Cys?Trp?Glu?Pro?Ala?Gln?Lys?Gly
130 135 140
Gly?Gly?Gly?Gly?Thr?Thr?Ser?Ala?Gly?Glu?Ser?Ala?Asp?Pro?Val?Thr
145 150 155 160
Ala?Thr?Val?Glu?Asn?Tyr?Gly?Gly?Glu?Thr?Gln?Val?Gln?Arg?Arg?Gln
165 170 175
His?Thr?Asp?Ile?Ala?Phe?Ile?Leu?Asp?Arg?Phe?Val?Lys?Val?Lys?Pro
180 185 190
Lys?Glu?Gln?Val?Asn?Val?Leu?Asp?Leu?Met?Gln?Ile?Pro?Ala?His?Thr
195 200 205
Leu?Val?Gly?Ala?Leu?Leu?Arg?Thr?Ala?Thr?Tyr?Tyr?Phe?Ser?Asp?Leu
210 215 220
Glu?Leu?Ala?Val?Lys?His?Glu?Gly?Asp?Leu?Thr?Trp?Val?Pro?Asn?Gly
225 230 235 240
Ala?Pro?Glu?Thr?Ala?Leu?Asp?Asn?Thr?Thr?Asn?Pro?Thr?Ala?Tyr?His
245 250 255
Lys?Glu?Pro?Leu?Thr?Arg?Leu?Ala?Leu?Pro?Tyr?Thr?Ala?Pro?His?Arg
260 265 270
Val?Leu?Ala?Thr?Val?Tyr?Asn?Gly?Ser?Ser?Lys?Tyr?Gly?Asp?Thr?Ser
275 280 285
Thr?Asn?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Glu
290 295 300
Arg?Thr?Leu?Pro?Thr?Ser?Phe?Asn?Phe?Gly?Ala?Ile?Lys?Ala?Thr?Arg
305 310 315 320
Val?Thr?Glu?Leu?Leu?Tyr?Arg?Met?Lys?Arg?Ala?Glu?Thr?Tyr?Cys?Pro
325 330 335
Arg?Pro?Leu?Leu?Ala?Ile?Gln?Pro?Ser?Asp?Ala?Arg?His?Lys?Gln?Arg
340 345 350
Ile?Val?Ala?Pro?Ala?Lys?Gln
355
<210>2
<211>382
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Met?Lys?Tyr?Thr?Ser?Tyr?Phe?Leu?Ala?Leu?Leu?Leu?Cys?Gly?Leu?Leu
1 5 10 15
Gly?Phe?Ser?Gly?Ser?Tyr?Gly?Gln?Gly?Gln?Phe?Phe?Arg?Glu?Ile?Glu
20 25 30
Asn?Leu?Lys?Glu?Tyr?Phe?Asn?Ala?Ser?Ser?Pro?Asp?Val?Ala?Lys?Gly
35 40 45
Gly?Pro?Leu?Phe?Ser?Glu?Ile?Leu?Lys?Asn?Trp?Lys?Asp?Glu?Ser?Asp
50 55 60
Lys?Lys?Ile?Ile?Gln?Ser?Gln?Ile?Val?Ser?Phe?Tyr?Phe?Lys?Leu?Phe
65 70 75 80
Glu?Asn?Leu?Lys?Asp?Asn?Gln?Val?Ile?Gln?Arg?Ser?Met?Asp?Ile?Ile
85 90 95
Lys?Gln?Asp?Met?Phe?Gln?Lys?Phe?Leu?Asn?Gly?Ser?Ser?Glu?Lys?Leu
100 105 110
Glu?Asp?Phe?Lys?Lys?Leu?Ile?Gln?Ile?Pro?Val?Asp?Asp?Leu?Gln?Ile
115 120 125
Gln?Arg?Lys?Ala?Ile?Asn?Glu?Leu?Ile?Lys?Val?Met?Asn?Asp?Leu?Ser
130 135 140
Pro?Lys?Ser?Asn?Leu?Arg?Lys?Arg?Lys?Arg?Ser?Gln?Asn?Leu?Phe?Arg
145 150 155 160
Gly?Arg?Arg?Ala?Ser?Thr?Gly?Gly?Gly?Gly?Gly?Thr?Thr?Ser?Ala?Gly
165 170 175
Glu?Ser?Ala?Asp?Pro?Val?Thr?Ala?Thr?Val?Glu?Asn?Tyr?Gly?Gly?Glu
180 185 190
Thr?Gln?Val?Gln?Arg?Arg?Gln?His?Thr?Asp?Ile?Ala?Phe?Ile?Leu?Asp
195 200 205
Arg?Phe?Val?Lys?Val?Lys?Pro?Lys?Glu?Gln?Val?Asn?Val?Leu?Asp?Leu
210 215 220
Met?Gln?Ile?Pro?Ala?His?Thr?Leu?Val?Gly?Ala?Leu?Leu?Arg?Thr?Ala
225 230 235 240
Thr?Tyr?Tyr?Phe?Ser?Asp?Leu?Glu?Leu?Ala?Val?Lys?His?Glu?Gly?Asp
245 250 255
Leu?Thr?Trp?Val?Pro?Asn?Gly?Ala?Pro?Glu?Thr?Ala?Leu?Asp?Asn?Thr
260 265 270
Thr?Asn?Pro?Thr?Ala?Tyr?His?Lys?Glu?Pro?Leu?Thr?Arg?Leu?Ala?Leu
275 280 285
Pro?Tyr?Thr?Ala?Pro?His?Arg?Val?Leu?Ala?Thr?Val?Tyr?Asn?Gly?Ser
290 295 300
Ser?Lys?Tyr?Gly?Asp?Thr?Ser?Thr?Asn?Asn?Val?Arg?Gly?Asp?Leu?Gln
305 310 315 320
Val?Leu?Ala?Gln?Lys?Ala?Glu?Arg?Thr?Leu?Pro?Thr?Ser?Phe?Asn?Phe
325 330 335
Gly?Ala?Ile?Lys?Ala?Thr?Arg?Val?Thr?Glu?Leu?Leu?Tyr?Arg?Met?Lys
340 345 350
Arg?Ala?Glu?Thr?Tyr?Cys?Pro?Arg?Pro?Leu?Leu?Ala?Ile?Gln?Pro?Ser
355 360 365
Asp?Ala?Arg?His?Lys?Gln?Arg?Ile?Val?Ala?Pro?Ala?Lys?Gln
370 375 380
<210>3
<211>1077
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
atgtggctgc?agaacctgct?tctcctgggc?actgtggtct?gcagcttctc?cgcacctact 60
cgcccaccca?acactgccac?ccggccctgg?cagcatgtgg?atgccatcaa?ggaggccctg 120
agccttctga?accacagcag?tgacactgat?gctgtgatga?atgacacaga?agtcgtctct 180
gaaaagtttg?actcccagga?accaacgtgc?ctgcagactc?gcctgaagct?gtacaagaac 240
ggcctgcagg?gcagcctcac?tagtctcatg?ggctccttga?ccatgatggc?cacccactac 300
gagaaacact?gcccacccac?cccggaaact?tcctgtggaa?cccagtttat?cagcttcaaa 360
aatttcaaag?aggacctgaa?ggagttcctt?tttatcattc?cctttgactg?ctgggaacca 420
gcccagaagg?gtggcggtgg?cggtaccacc?tctgcgggtg?agtctgcgga?ccccgtgact 480
gccaccgtcg?agaactacgg?tggtgagaca?caagtccaga?ggcgccagca?cacggacatt 540
gcgttcatac?tggacaggtt?cgtgaaagtc?aagccaaagg?aacaagttaa?tgtgttggac 600
ctgatgcaga?tccctgccca?caccttggta?ggggcgctcc?tgcgaacggc?cacctactac 660
ttctctgacc?tggagctggc?cgtcaagcac?gagggcgatc?tcacctgggt?cccaaacggc 720
gcccctgaga?cagcactgga?caacactacc?aacccaacag?cttaccacaa?ggaacccctc 780
acacggctgg?cgctgcctta?cacggctcca?caccgtgtct?tagcgaccgt?ctacaacggg 840
agcagtaagt?acggtgacac?cagcactaac?aacgtgagag?gtgaccttca?agtgttagct 900
cagaaggcag?aaagaactct?gcctacctcc?ttcaacttcg?gtgccatcaa?ggcaactcgt 960
gttactgaac?tactctacag?aatgaagaga?gccgagacat?actgtcccag?gccccttctc 1020
gccattcaac?cgagtgacgc?tagacacaag?cagaggattg?tggcacccgc?aaaacag 1077
<210>4
<211>1146
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
atgaaatata?caagctattt?cttagcttta?ctgctctgtg?ggcttttggg?tttttctggt 60
tcttatggcc?agggccaatt?ttttagagaa?atagaaaact?taaaggagta?ttttaatgca 120
agtagcccag?atgtagctaa?gggtgggcct?ctcttctcag?aaattttgaa?gaattggaaa 180
gatgaaagtg?acaaaaaaat?tattcagagc?caaattgtct?ccttctactt?caaactcttt 240
gaaaacctca?aagataacca?ggtcattcaa?aggagcatgg?atatcatcaa?gcaagacatg 300
tttcagaagt?tcttgaatgg?cagctctgag?aaactggagg?acttcaaaaa?gctgattcaa 360
attccggtgg?atgatctgca?gatccagcgc?aaagccataa?atgaactcat?caaagtgatg 420
aatgacctgt?caccaaaatc?taacctcaga?aagcggaaga?gaagtcagaa?tctctttcga 480
ggccggagag?catcaacggg?tggcggtggc?ggtaccacct?ctgcgggtga?gtctgcggac 540
cccgtgactg?ccaccgtcga?gaactacggt?ggtgagacac?aagtccagag?gcgccagcac 600
acggacattg?cgttcatact?ggacaggttc?gtgaaagtca?agccaaagga?acaagttaat 660
gtgttggacc?tgatgcagat?ccctgcccac?accttggtag?gggcgctcct?gcgaacggcc 720
acctactact?tctctgacct?ggagctggcc?gtcaagcacg?agggcgatct?cacctgggtc 780
ccaaacggcg?cccctgagac?agcactggac?aacactacca?acccaacagc?ttaccacaag 840
gaacccctca?cacggctggc?gctgccttac?acggctccac?accgtgtctt?agcgaccgtc 900
tacaacggga?gcagtaagta?cggtgacacc?agcactaaca?acgtgagagg?tgaccttcaa 960
gtgttagctc?agaaggcaga?aagaactctg?cctacctcct?tcaacttcgg?tgccatcaag 1020
gcaactcgtg?ttactgaact?actctacaga?atgaagagag?ccgagacata?ctgtcccagg 1080
ccccttctcg?ccattcaacc?gagtgacgct?agacacaagc?agaggattgt?ggcacccgca 1140
aaacag 1146
Claims (10)
1, a kind of fusion rotein of stimulating organism to produce FMDV antibody is made up of at least a cytokine among the VP1 albumen of foot and mouth disease virus and GM-CSF, the IFN-γ.
2, fusion rotein according to claim 1 is characterized in that: described cytokine is GM-CSF.
3, fusion rotein according to claim 1 is characterized in that: described fusion rotein has one of following amino acid residue sequences:
1) the SEQ ID NO:1 in the sequence table;
2) the SEQ ID NO:2 in the sequence table;
3) with the amino acid residue sequence of SEQ ID NO:1 in the sequence table or SEQ ID NO:2 through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of stimulating organism to produce FMDV antibody effect.
4, the gene of the described fusion rotein of coding claim 1 is one of following dna sequence dna:
1) the SEQ ID NO:3 in the sequence table;
2) the SEQ ID NO:4 in the sequence table;
3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO:3 in the sequence table or SEQ ID NO:4.
5, contain the described expression carrier of claim 4.
6, the transgenic cell line that contains the described gene of claim 4.
7, the engineering bacteria that contains the described gene of claim 4.
8, a kind of method of expressing the fusion rotein of the described stimulating organism to produce FMDV antibody of claim 1, be to make up the expression vector that contains the described fusion rotein encoding gene of claim 4, again the expression vector that makes up is imported host cell and obtain recombinant bacterial strain, cultivate this recombinant bacterial strain, from culture, isolate fusion rotein at last.
9, the application of the fusion rotein of the described stimulating organism to produce FMDV antibody of claim 1 in preventing and/or treating property of preparation aftosa vaccine.
10, the application of the fusion rotein encoding gene of the described stimulating organism to produce FMDV antibody of claim 4 in preventing and/or treating property of preparation aftosa vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101083394A CN100361708C (en) | 2005-10-12 | 2005-10-12 | Fusion protein for stimulating organism to produce FMDV antibody and its coding gene and uses |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101083394A CN100361708C (en) | 2005-10-12 | 2005-10-12 | Fusion protein for stimulating organism to produce FMDV antibody and its coding gene and uses |
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| Publication Number | Publication Date |
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| CN1763105A true CN1763105A (en) | 2006-04-26 |
| CN100361708C CN100361708C (en) | 2008-01-16 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB2005101083394A Expired - Fee Related CN100361708C (en) | 2005-10-12 | 2005-10-12 | Fusion protein for stimulating organism to produce FMDV antibody and its coding gene and uses |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210097077A (en) * | 2019-03-15 | 2021-08-06 | 대한민국(농림축산식품부 농림축산검역본부장) | An adjuvant which can be administered in combination with an oil emulsion for immunization of foot-and-mouth disease vaccine and vaccine composition containing the same adjuvant |
-
2005
- 2005-10-12 CN CNB2005101083394A patent/CN100361708C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210097077A (en) * | 2019-03-15 | 2021-08-06 | 대한민국(농림축산식품부 농림축산검역본부장) | An adjuvant which can be administered in combination with an oil emulsion for immunization of foot-and-mouth disease vaccine and vaccine composition containing the same adjuvant |
| KR102438048B1 (en) * | 2019-03-15 | 2022-08-31 | 대한민국 | An adjuvant which can be administered in combination with an oil emulsion for immunization of foot-and-mouth disease vaccine and vaccine composition containing the same adjuvant |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100361708C (en) | 2008-01-16 |
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