CN1749397A - Bi-recombinant active carrier vaccine of co-expression pig gyrate virus and foot and mouth disease virus - Google Patents
Bi-recombinant active carrier vaccine of co-expression pig gyrate virus and foot and mouth disease virus Download PDFInfo
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- CN1749397A CN1749397A CN 200410011047 CN200410011047A CN1749397A CN 1749397 A CN1749397 A CN 1749397A CN 200410011047 CN200410011047 CN 200410011047 CN 200410011047 A CN200410011047 A CN 200410011047A CN 1749397 A CN1749397 A CN 1749397A
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Abstract
The present invention belongs to the field of biotechnology, and is especially one kind of recombinant live carrier vaccine co-expressing pig gyrate virus and foot and mouth disease virus. The present invention aims at providing two recombinant chicken pox viruses to co-express capsid protein ORF2 or its fusion protein ORF2-ORF1 of the China epidemic strain of type-II pig gyrate virus and the structural protein precursor P1 of the China epidemic strain NY00 of type-O foot and mouth disease virus, and for serving as the duplex live carrier gene engineering vaccine to prevent PCV2 and FMDV infection. The two recombinant chicken pox viruses can stimulate mouse to generate effective body fluid immunity and cell immunity, and are safe to experimental animals without any pathological phenomenon.
Description
Technical field:
The invention belongs to biological technical field, particularly relate to the live recombinant vectors vaccine of a kind of coexpression pig circular ring virus and foot and mouth disease virus.
Background technology:
Pig circular ring virus (PCV) is the animal virus of a kind of minimum of finding so far.2 type pig circular ring virus (PCV2) have pathogenic, can cause pmws (PMWS), and it is a kind of new transmissible disease.Canada broke out this disease first in 1991, and the report that all there is this disease many in the world subsequently countries and regions causes serious economy loss to pig industry.Also having in its swinery of report in Jiangsu in China Taiwan, continent, Hebei etc. has PMWS to exist.PMWS brings sizable financial loss not only for the production of pig, and publilc health is also existed potential threatens, and when speaking of xenotransplantation especially, it is pathogenic former dangerous necessary assess the new pig that does not present characteristic.Therefore strengthen the PCV2 control, develop the severe problem that practicable PCV2 vaccine and medicine are researchers.
PCV belongs to PCV-II section in classification, PCV-II section is a section of (ICTV) the 6th academic report rebaptism in the ICTV, and the common trait of this coe virus is that a kind of little, icosahedron symmetry, no cyst membrane, sub-thread are circular DNA chain composition for virus.PCV is the self-replacation mammalian virus of the present minimum of finding, is made up of capsid protein and genome.The PCV-2 genome has 11 to read frames, i.e. ORF1 ~ ORF11, and each is read frame size and differs greatly, wherein ORF1, ORF5, ORF7, ORF10,5 ' ~ 3 ' direction is identical; ORF2, ORF3, ORF4, ORF6, ORF8, ORF9, ORF11,5 ' ~ 3 ' direction is identical.These reading frames show as overlapping genes, thereby utilize the limited genetic material of PCV-II as far as possible economically, and this may be the result of natural selection in the organic evolution process.
PCV-2ORF1 has 945 bases, 314 amino acid of encoding, and PCV-2ORF2 has 702 bases, 234 amino acid of encoding.Infer that from aminoacid sequence ORF1 has 3 glycosylation sites, ORF2 and ORF5 respectively have 1 glycosylation site.Read in the frames at 11, study more deep be ORF1 and ORF2, and present research to ORF2 has become the focus that PCV2 studies.Liu Q etc. has studied the location of PCV-2ORF2 in cell with immunofluorescence method, find that 41 amino acid of ORF2 albumen n end are basic aminoacids mostly, and sequence is more conservative.Further analysis revealed the 12nd is to the 18th, and the 34th to the 41st location of amino acid domain in nuclear plays an important role.
Mahe D etc. with rhabdovirus system expression ORF1 and ORF2, though find that PCV-1ORF2 is relevant with the PCV-2ORF2 antigenicity, have only the ORF2 albumen can be by PCV2 anti-identification how.Lir Q etc. are connected to MBP-His (8)-tag gene 3 ' end with the ORF2 gene, have expressed the ORF2 fusion rotein in bacterium, this fusion rotein can with the how anti-reaction of PCV2.This shows and can distinguish PCV-1 and PCV-2 with ORF2.The inoculation of usefulness such as TruongC has the antiserum(antisera) of collecting in the PCV2 swinery to do experiment, detects (ELISA) by enzyme linked immunological absorption and identifies PCV2-ORF2 proteins encoded and Ia B cell linear epitope.Also utilize the ORF2 epitope to learn the sign that detects as PCV test pig and natural infection porcine blood serum.Also have two virus O RF1 of report proteins encoded such as Dominique Mahe that the antigen dependency is arranged, yet their ORF2 encoded protein is but resisted the identification difference of PCV2 polyclonal antibody.And PEPSCAN analyzes the overlapping fragments of ORF1, ORF2, ORF3 encoding part gene, found five with Ia determining area, one of them is positioned at ORF1, and in addition four be arranged in ORF2.And find that only some ORF2 polypeptide are immune associated epitope of difference virus type.Thereby hint uses the antigen that comes from ORF2 as diagnostic tool very big potential to be arranged.Just because of the above characteristic of ORF2, make it become the desirable antigen of diagnosis PCV on the molecular level clinically.Therefore, ORF2 also will bring into play the big effect of providing in the PCV-2 serosurvey.
Up to the present not fully aware of about this sick pathogenesis.Allan etc. think should pay attention to PCV-2 and the acting in conjunction of pig picornavirus.Because the pig picornavirus is bred in the immunocyte of pig, brings out immunosuppression probably, promote the propagation of PCV-2, perhaps the pig picornavirus has activated the susceptibility to PCV-2.
The no effective methods of treatment of this disease does not have effective vaccine both at home and abroad and can utilize.Since Wloff etc. found this new technique of naked DNA, many researchs had shown that DNA can induce protective immunological reaction to resist virus in the animal model.The plasmid DNA of also finding injection coding Pseudorabies virus glycoprotein in the past in the investigation can produce aversion response opposing lethal infection in pig muscle; Collaborative coding porcine interleukin can improve immune response, produces a kind of immunological adjuvant effect.So this research will make up nucleic acid vaccine and live vector recombinant viral genome engineering seedling,, lay the foundation for further developing efficient, safe new generation vaccine in the hope of obtaining the desirable vaccine of prevention PCV2.
The genome of foot and mouth disease virus is a single-stranded RNA, is made up of about 8500 Nucleotide, and its molecular weight is about 2.6 * 10
6Da can be divided into 5 ' end non-coding region, P1 district, P2 district, P3 district and 3 ' end non-coding region.Main structural protein of P1 district coding wherein, P2 district and P3 district coding and virus replication, translation, and complete virus particle assembles relevant Nonstructural Protein.
Structural protein P1 region nucleotide sequence total length is 2154nt, is made up of 1A, 1B, 1C and 4 gene coded sequences of 1D, and length is followed successively by 167,648,660 and 639nt, 85,218,220 and 213 the amino acid whose structural polypeptides of encoding respectively.1B, 1C and 1D are positioned at the capsid surface, are outer capsid albumen.1D encoding histone polypeptide VP1 wherein, its major part is exposed to virus surface, is the main component of decision virus antigenicity.But other three kinds of structural polypeptides also all participate in immunogenic formation.The antigen site of different serotypes virus is different, and the O C-type virus C has 5 sites: antigen site 1 is made up of 1D GH ring (1133-1157) and 200-213 amino acid whose linear epitope.This site is the topmost antigen site of FMDV, also is the key point of FMDV antigen site.
(Fowlpox Virus, FPV) expression vector system is another animal virus vector that is similar to the canary pox virus carrier to bird pox virus.FPV has the advantage identical with the canary pox virus carrier as carrier, and in addition, than the canary pox virus carrier, its genome structure is more huge, can hold bigger foreign gene and does not lose its infectivity; By the foreign protein of being expressed, in cells infected, can verily modify, as glycosylation, carboxylated etc.; The expression of exogenous gene product has good immunogenicity, can induce body to produce long-term cellular immunization and humoral immunization; Duplicate in the strict endochylema, avoided virogene to be recombined into the possibility of host cell chromosome, eliminated recombinant virus and used the potential threat of back people and animals.The host specificity of FPV makes reorganization FPV (Recombinant FPV; rFPV) only produce abortive infection behind the seeded with mammalian; but still can be in the animal of wider range correct expression alien gene; produce the correct antigen of structure picture at surface of cell membrane; thereby bring out body and produce protective immunity; and safety, effective, especially the animal to immune deficiency and immunocompromised is even more ideal carrier.So FPV not only can be used to develop the genetically engineered living vaccine of bird, and can be used as the non-replicating virus vector, development mammalian genes engineering living vaccine is used for bird animal and even human immunity in addition.Therefore, the research of FPV carrier, especially FPV are subject to people's attention day by day as the research of nonreplication vector.
Summary of the invention:
The object of the present invention is to provide the recombinant Borrel virus of two kinds of difference coexpression 2 type pig circular ring virus (PCV2) Chinese epidemic strain capsid protein ORF2 or ORF2-ORF1 (replication protein) fusion rotein and O type foot and mouth disease virus (FMDV) Chinese epidemic strain NY00 strain structural protein precursor P1, as the bigeminy genetically engineered live vector vaccine of prevention China PCV2 and FMDV infection.
The present invention sets up a new pig circular ring virus genomic library: the application PCR method increases the full gene segmentation of Chinese PCR2 epidemic strain nm2002 and is cloned in pMD18-T and the pGEM-T Easy carrier, two plasmids of pMD18T-ORF2 and pGEMT-XS have been made up, splicing and obtain the whole genome sequence of Chinese PCV2 epidemic strain nm2002;
Again gene involved in immunity is cloned into the expression vector from the Central Asia, DNA library that makes up, in host cell, gives expression to corresponding gene, as prevention or to examine protein, genetic recombinants or the recombinant immune of pmws former.
The present invention is inserted into bird pox virus expression vector pUTAL P7.5 tandem promoter downstream from the pKS-P1 carrier after with HindIII and SalI double digestion with the P1 district gene of FMDV O-NY00 strain, form intermediate carrier pUTALP1, after simultaneously the PCV2 ORF2 gene of Chinese epidemic strain PCV2nm2002 being used HincII and SmaI double digestion from carrier pMD18T-ORF2, be inserted into bird pox virus expression vector pUTALP1ATI-P7.5 combined promoter downstream, made up coexpression PCV2ORF2 and FMDV O-NY00 P1 recombinant Borrel transferring plasmid.
The gene order of pUTALP1-ORF2 is:
atg?acg?tat?cca?agg?agg?cgt?tac?cgg?aga?aga?aga?cac?cgc?ccc?cgc 48
Met?Thr?Tyr?Pro?Arg?Arg?Arg?Tyr?Arg?Arg?Arg?Arg?His?Arg?Pro?Arg
1 5 10 15
agc?cat?ctt?ggc?cag?atc?ctc?cgc?cgc?cgc?ccc?tgg?ctc?gtc?cac?ccc 96
Ser?His?Leu?Gly?Gln?Ile?Leu?Arg?Arg?Arg?Pro?Trp?Leu?Val?His?Pro
20 25 30
cgc?cac?cgt?tac?cgc?tgg?aga?agg?aaa?aat?ggc?atc?ttc?aac?acc?cgc 144
Arg?His?Arg?Tyr?Arg?Trp?Arg?Arg?Lys?Asn?Gly?Ile?Phe?Asn?Thr?Arg
35 40 45
ctc?tcc?cgc?acc?ttc?gga?tat?act?atc?aag?cga?acc?aca?gtc?aaa?acg 192
Leu?Ser?Arg?Thr?Phe?Gly?Tyr?Thr?Ile?Lys?Arg?Thr?Thr?Val?Lys?Thr
50 55 60
ccc?tcc?tgg?gcg?gtg?gac?atg?atg?aca?ttc?aat?att?aat?gac?ttt?ctt 240
Pro?Ser?Trp?Ala?Val?Asp?Met?Met?Thr?Phe?Asn?Ile?Asn?Asp?Phe?Leu
65 70 75 80
ccc?cca?gga?ggg?ggc?tca?aac?ccc?cgc?tct?gtg?ccc?ttt?gaa?tac?tac 288
Pro?Pro?Gly?Gly?Gly?Ser?Asn?Pro?Arg?Ser?Val?Pro?Phe?Glu?Tyr?Tyr
85 90 95
aga?ata?aga?aag?gtt?aag?gtt?gaa?ttc?tgg?ccc?tgc?tcc?ccg?atc?acc 336
Arg?Ile?Arg?Lys?Val?Lys?Val?Glu?Phe?Trp?Pro?Cys?Ser?Pro?I1e?Thr
100 105 110
cag?ggt?gac?agg?gga?gtg?ggc?tcc?agt?gct?gtt?att?cta?gat?gat?aac 384
Gln?Gly?Asp?Arg?Gly?Val?Gly?Ser?Ser?Ala?Val?Ile?Leu?Asp?Asp?Asn
115 120 125
ttt?gta?aca?aag?gcc?aca?gcc?ctc?acc?tat?gac?ccc?tat?gta?aac?tac 432
Phe?Val?Thr?Lys?Ala?Thr?Ala?Leu?Thr?Tyr?Asp?Pro?Tyr?Val?Asn?Tyr
130 135 140
tcc?tcc?cgc?cat?acc?ata?acc?cag?ccc?ttc?tcc?tac?cac?tcc?cgc?tac 480
Ser?Ser?Arg?His?Thr?Ile?Thr?Gln?Pro?Phe?Ser?Tyr?His?Ser?Arg?Tyr
145 150 155 160
ttt?acc?ccc?aaa?cct?gtc?cta?gat?tcc?act?att?gat?tac?ttc?caa?cca 528
Phe?Thr?Pro?Lys?Pro?Val?Leu?Asp?Ser?Thr?Ile?Asp?Tyr?Phe?Gln?Pro
165 170 175
aac?aac?aaa?aga?aat?cag?ctg?tgg?ctg?aga?cta?caa?act?gct?gga?aat 576
Ash?Asn?Lys?Arg?Asn?Gln?Leu?Trp?Leu?Arg?Leu?Gln?Thr?Ala?Gly?Asn
180 185 190
gta?gac?cac?gta?ggc?ctc?ggc?act?gcg?ttc?gaa?aac?agt?ata?tac?gac 624
Val?Asp?His?Val?Gly?Leu?Gly?Thr?Ala?Phe?Glu?Asn?Ser?Ile?Tyr?Asp
195 200 205
cag?gaa?tac?aat?atc?cgt?gta?acc?atg?tat?gta?caa?ttc?aga?gaa?ttt 672
Gln?Glu?Tyr?Asn?Ile?Arg?Val?Thr?Met?Tyr?Val?Gln?Phe?Arg?Glu?Phe
210 215 220
aat?ctt?aaa?gac?ccc?cca?ctt?aac?ccc?taa 702
Asn?Leu?Lys?Asp?Pro?Pro?Leu?Asn?Pro
225 230
The present invention is inserted into bird pox virus expression vector pUTAL P7.5 tandem promoter downstream from the pKS-P1 carrier after with HindIII and SalI double digestion with the P1 district gene of FMDV O-NY00 strain, form intermediate carrier pUTALP1, simultaneously with the middle interstitial granules pMD18T-ORF2-ORF1 of the ORF2-ORF1 fusion gene of Chinese epidemic strain PCV2nm2002 with HincII and SmaI double digestion after, be inserted into bird pox virus expression vector pUTALATI-P7.5 combined promoter downstream, make up coexpression PCV2ORF2-ORF1 and FMDV O-NY00 P1 recombinant Borrel transferring plasmid pUTALP1-ORF2-ORF1.
The gene order of pUTALPl-ORF2-ORF1 is:
atg?acg?tat?cca?agg?agg?cgt?tac?cgg?aga?aga?aga?cac?cgc?ccc?cgc 48
Met?Thr?Tyr?Pro?Arg?Arg?Arg?Tyr?Arg?Arg?Arg?Arg?His?Arg?Pro?Arg
1 5 10 15
agc?cat?ctt?ggc?cag?atc?ctc?cgc?cgc?cgc?ccc?tgg?ctc?gtc?cac?ccc 96
Ser?His?Leu?Gly?Gln?Ile?Leu?Arg?Arg?Arg?Pro?Trp?Leu?Val?His?Pro
20 25 30
cgc?cac?cgt?tac?cgc?tgg?aga?agg?aaa?aat?ggc?atc?ttc?aac?acc?cgc 144
Arg?His?Arg?Tyr?Arg?Trp?Arg?Arg?Lys?Asn?Gly?Ile?Phe?Asn?Thr?Arg
35 40 45
ctc?tcc?cgc?acc?ttc?gga?tat?act?atc?aag?cga?acc?aca?gte?aaa?acg 192
Leu?Ser?Arg?Thr?Phe?Gly?Tyr?Thr?Ile?Lys?Arg?Thr?Thr?Val?Lys?Thr
50 55 60
ccc?tcc?tgg?gcg?gtg?gac?atg?atg?aca?ttc?aat?att?aat?gac?ttt?ctt 240
Pro?Ser?Trp?Ala?Val?Asp?Met?Met?Thr?Phe?Asn?Ile?Asn?Asp?Phe?Leu
65 70 75 80
ccc?cca?gga?ggg?ggc?tca?aac?ccc?cgc?tct?gtg?ccc?ttt?gaa?tac?tac 288
Pro?Pro?Gly?Gly?Gly?Ser?Asn?Pro?Arg?Ser?Val?Pro?Phe?Glu?Tyr?Tyr
85 90 95
aga?ata?aga?aag?gtt?aag?gtt?gaa?ttc?tgg?ccc?tgc?tcc?ccg?atc?acc 336
Arg?Ile?Arg?Lys?Val?Lys?Val?Glu?Phe?Trp?Pro?Cys?Ser?Pro?Ile?Thr
100 105 110
cag?ggt?gac?agg?gga?gtg?ggc?tcc?agt?gct?gtt?att?cta?gat?gat?aac 384
Gln?Gly?Asp?Arg?Gly?Val?Gly?Ser?Ser?Ala?Val?Ile?Leu?Asp?Asp?Asn
115 120 125
ttt?gta?aca?aag?gcc?aca?gcc?ctc?acc?tat?gac?ccc?tat?gta?aac?tac 432
Phe?Val?Thr?Lys?Ala?Thr?Ala?Leu?Thr?Tyr?Asp?Pro?Tyr?Val?Asn?Tyr
130 135 140
tcc?tcc?cgc?cat?acc?ata?acc?cag?ccc?ttc?tcc?tac?cac?tcc?cgc?tac 480
Ser?Ser?Arg?His?Thr?Ile?Thr?Gln?Pro?Phe?Ser?Tyr?His?Ser?Arg?Tyr
145 150 155 160
ttt?acc?ccc?aaa?cct?gtc?cta?gat?tcc?act?att?gat?tac?ttc?caa?cca 528
Phe?Thr?Pro?Lys?Pro?Val?Leu?Asp?Ser?Thr?Ile?Asp?Tyr?Phe?Gln?Pro
165 170 175
aac?aac?aaa?aga?aat?cag?ctg?tgg?ctg?aga?cta?caa?act?gct?gga?aat 576
Asn?Asn?Lys?Arg?Asn?Gln?Leu?Trp?Leu?Arg?Leu?Gln?Thr?Ala?Gly?Asn
180 185 190
gta?gac?cac?gta?ggc?ctc?ggc?act?gcg?ttc?gaa?aac?agt?ata?tac?gac 624
Val?Asp?His?Val?Gly?Leu?Gly?Thr?Ala?Phe?Glu?Asn?Ser?Ile?Tyr?Asp
195 200 205
cag?gaa?tac?aat?atc?cgt?gta?acc?atg?tat?gta?caa?ttc?aga?gaa?ttt 672
Gln?Glu?Tyr?Asn?Ile?Arg?Val?Thr?Met?Tyr?Val?Gln?Phe?Arg?Glu?Phe
210 215 220
aat?ctt?aaa?gac?ccc?cca?ctt?aac?cct?tat?act?agt?ctg?aaa?acg?aaa 720
Asn?Leu?Lys?Asp?Pro?Pro?Leu?Asn?Pro?Tyr?Thr?Ser?Leu?Lys?Thr?Lys
225 230 235 240
gaa?gtg?cgc?tgt?aag?tat?tac?cag?cgc?act tcg?gca?gcg?gca?gca?cct 768
Glu?Val?Arg?Cys?Lys?Tyr?Tyr?Gln?Arg?Thr?Ser?Ala?Ala?Ala?Ala?Pro
245 250 255
cgg?cag?cac?ctc?agc?agc?aac?atg?ccc?agc?aag?aag?aat?gga?aga?agc 816
Arg?Gln?His?Leu?Ser?Ser?Asn?Met?Pro?Ser?Lys?Lys?Asn?Gly?Arg?Ser
260 265 270
gga?ccc?caa?ccc?cat?aaa?agg?tgg?gtg?ttc?act?ctg?aat?aat?cct?tcc 864
Gly?Pro?Gln?Pro?His?Lys?Arg?Trp?Val?Phe?Thr?Leu?Asn?Asn?Pro?Ser
275 280 285
gaa?gac?gag?cgc?aag?aaa?ata?cgg?gat?ctt?cca?ata?tcc?cta?ttt?gat 912
Glu?Asp?Glu?Arg?Lys?Lys?Ile?Arg?Asp?Leu?Pro?Ile?Ser?Leu?Phe?Asp
290 295 300
tat?ttt?att?gtt?ggc?gag?gag?ggt?aat?gag?gaa?gga?cga?aca?cct?cac 960
Tyr?Phe?Ile?Val?Gly?Glu?Glu?Gly?Asn?Glu?Glu?Gly?Arg?Thr?Pro?His
305 310 315 320
ctc?cag?ggg?ttc?gct?aat?ttt?gtg?aag?aag?cag?act?ttt?aat?aaa?gtg 1008
Leu?Gln?Gly?Phe?Ala?Asn?Phe?Val?Lys?Lys?Gln?Thr?Phe?Asn?Lys?Val
325 330 335
aag?tgg?tat?ttg?ggt?gcc?cgc?tgc?cac?atc?gag?aaa?gcg?aaa?gga?aca 1056
Lys?Trp?Tyr?Leu?Gly?Ala?Arg?Cys?His?Ile?Glu?Lys?Ala?Lys?Gly?Thr
340 345 350
gat?cag?cag?aat?aaa?gaa?tac?tgc?agt?aaa?gaa?ggc?aac?tta?ctg?atg 1104
Asp?Gln?Gln?Asn?Lys?Glu?Tyr?Cys?Ser?Lys?Glu?Gly?Asn?Leu?Leu?Met
355 360 365
gag?tgt?gga?gct?cct?aga?tct?cag?gga?caa?cgg?agt?gac?ctg?tct?act 1152
Glu?Cys?Gly?Ala?Pro?Arg?Ser?Gln?Gly?Gln?Arg?Ser?Asp?Leu?Ser?Thr
370 375 380
gct?gtg?agt?acc?ttg?ttg?gag?agc?ggg?agt?ctg?gtg?acc?gtt?gca?gag 1200
Ala?Val?Ser?Thr?Leu?Leu?Glu?Ser?Gly?Ser?Leu?Val?Thr?Val?Ala?Glu
385 390 395 400
cag?cac?cct?gta?acg?ttt?gtc?aga?aat?ttc?cgc?ggg?ctg?gct?gaa?ctt 1248
Gln?His?Pro?Val?Thr?Phe?Val?Arg?Asn?Phe?Arg?Gly?Leu?Ala?Glu?Leu
405 410 415
ttg?aaa?gtg?agc?ggg?aaa?atg?cag?aag?cgt?gat?tgg?aag?act?aat?gta 1296
Leu?Lys?Val?Ser?Gly?Lys?Met?Gln?Lys?Arg?Asp?Trp?Lys?Thr?Asn?Val
420 425 430
cac?gtc?att?gtg?ggg?cca?cct?ggg?tgt?ggt?aaa?agc?aaa?tgg?gct?gct 1344
His?Val?Ile?Val?Gly?Pro?Pro?Gly?Cys?Gly?Lys?Ser?Lys?Trp?Ala?Ala
435 440 445
aat?ttt?gca?gac?ccg?gaa?acc?aca?tac?tgg?aaa?cca?cct?aga?aac?aag 1392
Asn?Phe?Ala?Asp?Pro?Glu?Thr?Thr?Tyr?Trp?Lys?Pro?Pro?Arg?Asn?Lys
450 455 460
tgg?tgg?gat?ggt?tac?cat?ggt?gaa?gaa?gtg?gtt?gtt?att?gat?gac?ttt 1440
Trp?Trp?Asp?Gly?Tyr?His?Gly?Glu?Glu?Val?Val?Val?Ile?Asp?Asp?Phe
465 470 475 480
tat?ggc?tgg?ctg?ccc?tgg?gat?gat?cta?ctg?aga?ctg?tgt?gat?cga?tat 1488
Tyr?Gly?Trp?Leu?Pro?Trp?Asp?Asp?Leu?Leu?Arg?Leu?Cys?Asp?Arg?Tyr
485 490 495
cca?ttg?act?gta?gag?act?aaa?ggt?gga?act?gta?cct?ttt?ttg?gcc?cgc 1536
Pro?Leu?Thr?Val?Glu?Thr?Lys?Gly?Gly?Thr?Val?Pro?Phe?Leu?Ala?Arg
500 505 510
agt?att?ctg?att?acc?agc?aat?cag?acc?ccg?ttg?gaa?tgg?tac?tcc?tca 1584
Ser?Ile?Leu?Ile?Thr?Ser?Asn?Gln?Thr?Pro?Leu?Glu?Trp?Tyr?Ser?Ser
515 520 525
act?gct?gtc?cca?gct?gta?gaa?gct?ctt?tat?cgg?agg?att?act?tcc?ttg 1632
Thr?Ala?Val?Pro?Ala?Val?Glu?Ala?Leu?Tyr?Arg?Arg?Ile?Thr?Ser?Leu
530 535 540
gta?ttt?tgg?aag?aat?gct?aca?gaa?caa?tcc?acg?gag?gaa?ggg?ggc?cag 1680
Val?Phe?Trp?Lys?Asn?Ala?Thr?Glu?Gln?Ser?Thr?Glu?Glu?Gly?Gly?Gln
545 550 555 560
ttc?gtc?acc?ctt?tcc?ccc?cca?tgc?cct?gaa?ttt?cca?tat?gaa?ata?aat 1728
Phe?Val?Thr?Leu?Ser?Pro?Pro?Cys?Pro?Glu?Phe?Pro?Tyr?Glu?Ile?Asn
565 570 575
tac?tga 1734
Tyr
The associated protein of indication of the present invention can obtain expressing in the plant rhabdovirus expression vector from insect cell, also can be animal virus expression vector or the expression of other carrier for expression of eukaryon in mammalian cell.
Advantage of the present invention and positively effect:
1, the two strain recombinant Borrel virus that obtain of the present invention can be expressed PCV2 capsid protein and FMDV P1 structural protein simultaneously, and expressed proteins can be respectively and corresponding antibody generation specific reaction.
2, the two strain recombinant Borrel virus that obtain of the present invention can stimulate mouse to produce effective humoral immunization and cellular immunization.
3, the two strain recombinant Borrel virus that obtain of the present invention are safe to laboratory animal, do not have any pathological phenomena.
4, the two strain recombinant Borrel virus that obtain of the present invention have good genetic stability, and foreign gene is not lost after repeatedly going down to posterity, and can express foreign protein in non-poultry animal (as Mammals) cell.
5, goal gene used in the present invention is the P1 gene of Chinese epidemic strain O_NY00 strain FMDV and PCV2 capsid protein ORF2, replication protein ORF1 gene, thereby the recombinant Borrel virus that makes up can be used as the bigeminy genetically engineered live vector vaccine of prevention 2 type pig circular ring virus and O type foot and mouth disease virus.
Description of drawings:
Fig. 1 is that recombinant plasmid pUTALP1-ORF2 of the present invention makes up schema;
Fig. 2 is that recombinant plasmid pUTALP1-ORF2-ORF1 of the present invention makes up schema.
Embodiment:
1. the screening of being connected of the recovery of the extraction of the preparation of competent escherichia coli cell and conversion, plasmid and digestion with restriction enzyme, dna fragmentation, linear DNA fragment, recombinant plasmid and evaluation, pcr amplification reaction etc. are translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., and Science Press (1992) related Sections carries out.
1.1 the preparation of competent escherichia coli cell
Under aseptic condition, picking is frozen in the bacterial classification of-70 ℃ of refrigerators, and streak inoculation is inverted for 37 ℃ and is cultivated about 16h on the LB agar plate that does not contain penbritin (Amp).The single bacterium colony of picking is inoculated into 3ml and does not contain in the antibiotic LB nutrient solution 37 ℃ of jolting overnight incubation.Aseptic absorption next day 1ml inoculum joins the 100ml that is preheated to 37 ℃ and does not contain in the LB nutrient solution of Amp, and 37 ℃ of violent joltings (200r/min) are cultivated.Be cultured to OD
600=0.4～0.5 (after about 2.5～3h), ice bath 15min makes culture be cooled to 0 ℃.Under aseptic condition, (notice that below operation all needs strict aseptic and carry out in ice-water bath), inoculum transferred in the 50ml polypropylene centrifuge tube of 2 aseptic and ice precoolings that centrifugal (4000r/min) 10min abandons supernatant under 4 ℃ of conditions; Reclaim bacterial precipitation, every pipe adds the 75mmol/LCaCl of 10ml ice precooling
2, the resuspended precipitation of 10mmol/L TrisCl (pH6.5) solution, ice bath 10min, 4 ℃ of centrifugal 10min of 4000r/min thoroughly abandon supernatant; The cold CaCl of every effective 2ml ice bath
2Preserve liquid [75mmol/L CaCl
2, 10mmol/L TrisCl pH6.5,15% (v/v) glycerine] bacterial sediment is hanged.Resuspended good competent cell is sub-packed in the aseptic Eppendorf tube, and every pipe 100l puts-70 ℃ of Ultralow Temperature Freezers or frozen standby in liquid nitrogen.
1.2 the connection of PCR product
(pMD18-T, pGEM-T Easy T carrier are connected, and (pGEM-Teasy Vector Kit, Promega company) illustrates and operate according to test kit with the T carrier with the PCR product.T carrier (30ng/l) 2l, T4 ligase enzyme 1l, it is an amount of and add water and mend to 10l to reclaim dna fragmentation, in 4 ℃ of reaction 14～16h.
1.3 transform
In frozen water, melt a competent cell, 2l is connected that product adds wherein and mixing gently, ice bath 30min, behind 42 ℃ of thermal shocking 90s, ice bath 10min adds 250l LB substratum (room temperature condition) immediately, 37 ℃ of jolting 225r/min 1h.Get 50l and 200l and be added to LB (containing 25g/ml kana or the 50g/ml Amp) agar plate that scribbles a certain amount of X-Gal (50mg/ml) and IPTG (200mg/ml) in advance, evenly after the coating, treat that bacterium liquid blots after, place 37 ℃ of overnight incubation.
1.4 the extraction of plasmid
1.4.1 a small amount of of plasmid preparation (alkaline lysis)
Under aseptic condition, with the single conversion bacterium colony of aseptic toothpick picking, being inoculated in 3ml contains in the corresponding antibiotic LB liquid nutrient medium, 37 ℃ of 250r/min jolting overnight incubation, culture is changed in the Eppendorf tube of 1.5ml, the centrifugal 3min of 6000r/min is inverted in pipe on the thieving paper, inhales as much as possible and removes residual nutrient solution.The resuspended bacterial precipitation of solution I (50mmol/L glucose, 25mmol/L TrisCl pH8.0,10mmol/L EDTA) with the precooling of 100l ice.(0.2mol/L NaOH 1%SDS), puts upside down mixing for several times, adds the solution III (3mol/L KAC, 5mol/L glacial acetic acid) of 150l ice precooling, puts upside down mixing, ice bath 5min, 4 ℃ of centrifugal 10min of 12000r/min to add the new solution II of preparing of 200l.Get supernatant and add the saturated phenol of equivalent respectively, saturated phenol: chloroform: primary isoamyl alcohol (25: 24: 1), chloroform: each extracting of primary isoamyl alcohol (24: 1) once, get at last and reset and add 2 times of cold dehydrated alcohols of volume and 1/10 volume 3M NaAc, mix, place more than the 2h for 0 ℃, the centrifugal 10min of 12000r/min, abandon supernatant, to precipitate with twice of 70% cold washing with alcohol (noting precipitating not washed losing), drying at room temperature, TE (10mmol/L TrisCl pH8.0, the 1mmol/L EDTA) dissolution precipitation of RNA enzyme (10mg/ml) that contains the no DNA enzyme of final concentration 20g/ml with 20l, 37 ℃ of water-bath 30min, after agarose gel electrophoresis was identified, it was standby to put-20 ℃ of preservations.
1.4.2 a large amount of extraction and purifications of plasmid
The microbionation that will contain the purpose plasmid is in the LB of 250ml substratum (containing corresponding microbiotic), 16～18h is cultivated in 37 ℃ of 200r/min joltings, and 4 ℃ of centrifugal 10min of 4000r/min abandon supernatant, precipitation is iced STE (the 0.1mmol/L NaCl of precooling with 50ml, 10mmol/L TrisCl pH8.0,50mmol/L EDTA) resuspended precipitation, 4 ℃ of centrifugal 10min of 4000r/min, abandon supernatant, precipitation adds the new solution II of preparing of 10ml, fully mixing with the resuspended precipitation of solution I of 5ml ice precooling, the solution III that adds the precooling of 7.5ml ice, mixing gently, ice bath 10～15min, 4 ℃ of centrifugal 15min of 7000r/min, supernatant through 4 layers of filtered through gauze to 50ml in the aseptic centrifuge tube, add 0.6 times of volume Virahol, fully mixing is placed 30min in the room temperature, the centrifugal 10min of 10000r/min, abandon supernatant, precipitation is washed once drying with 70% ethanol.After adding an amount of TE (pH8.0) dissolution precipitation, add equal-volume 5mol/L LiCl, abundant mixing, ice bath is placed 10min, the centrifugal 10min of 12000r/min, get supernatant, add the abundant mixing of equivalent Virahol, the centrifugal 10min of 10000r/min, abandon supernatant, use 70% washing with alcohol, drying at room temperature, with an amount of TE (pH8.0) dissolution precipitation, the RNA enzyme (10mg/ml) that adds no DNA enzyme is to final concentration 20g/ml, 37 ℃ of water-bath 30min, add the 1.6mol/L NaCl that equal-volume contains 13% (w/v) polyglycol solution (PEG8000), fully mixing is put 4 ℃ and is spent the night, 4 ℃ of centrifugal 10min of 12000r/min, abandon supernatant, add 400l TE (pH8.0) dissolution precipitation, with the saturated phenol of equal-volume, phenol: chloroform: primary isoamyl alcohol (25: 24: 1), chloroform: each extracting of primary isoamyl alcohol (24: 1) once, add 100l NaAC, 2 times of cold dehydrated alcohols of volume, mixing postposition-20 ℃ is deposited more than the 30min 4 ℃ of centrifugal 10min of 12000r/min, abandon supernatant, precipitate with 70% cold washing with alcohol, drying at room temperature is dissolved among the TE (pH8.0), calculate plasmid DNA concentration, be stored in-20 ℃.
1.5 plasmid DNA is quantitative
With TE (pH8.0) is blank, measures nucleic acid solution optical density(OD) (OD) value of wavelength 260nm and 280nm with TZK-800Z type ultraviolet spectrophotometer.OD
260=l is equivalent to contain the about 50g/ml of plasmid, the OD of the pure product of double-stranded DNA
260/ OD
280Value is 1.8, if OD
260/ OD
280Value is starkly lower than 1.8, then has protein or phenol and pollutes, and need be further purified.
1.6 the digestion of restriction enzyme
Single enzyme endonuclease reaction is with plasmid DNA and the suitable quantity of water mixing of 1.0g, making its cumulative volume is 20l, add 2～3ul restriction enzyme and the corresponding 10 * restriction enzyme reaction damping fluid of 2l, flick tube wall mixing and centrifugal, put optimal reactive temperature water-bath 2～3h, get the 5l reaction solution and carry out the agarose gel electrophoresis inspection.To the endonuclease reaction of a large amount of plasmid DNA, corresponding expansion restriction enzyme enzyme dosage and reaction volume, the reaction solution electrophoretic examinations that takes a morsel is behind the complete degestion, in-20 ℃ of preservations, in order to further identifying or reclaim the usefulness of fragment.
Two enzyme endonuclease reactions select two kinds of activity of enzyme reaction all to carry out double digestion reaction, the same single endonuclease digestion of the concrete grammar of its reaction in 75%～100% same buffering system.If temperature or buffering system difference, then by high temperature behind the first low temperature, the order of high salt is carried out behind the first less salt.Or first after enzyme cuts, and behind phenol, the imitative extracting DNA, ethanol sedimentation carries out second endonuclease reaction again.
1.7 from pathological material of disease, extract DNA
1) will the die of illness internal organs multigelation three times of pig is got 2g inguinal lymph nodes or mesenteric lymph nodes and is fully ground, and grinds to make it become tissue homogenate.
2) 37 ℃ of water-baths are 1 hour, and homogenate places centrifuge tube.
3) 5000rpm is centrifugal 10 minutes, gets supernatant liquor, adds 300ug/mL Proteinase K and 1%SDS therein, and 56 ℃ digested 2 hours, and used phenol, chloroform extracting 2-3 time again.
4) carefully suct clearly in clean centrifuge tube, add the NaAC of 1/10 volume and the dehydrated alcohol of 2 times of volumes.
5) 12000rpm is centrifugal 15 minutes, and precipitation is dissolved in an amount of TE damping fluid, and-20 ℃ of preservations are standby.1.8PCR the PCR reaction solution is at first disposed in reaction, comprises 10 * Ex Taq Buffer (no Mg
2+), MgCl
2(25mM), Ex Taq
TMArchaeal dna polymerase, upstream primer (20pM), downstream primer (20pM) and deionized water mix by suitable component, add above-mentioned dna profiling product again.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min; 51 ℃ of annealing 1min; 72 ℃ are extended 1min; After 30 circulations, 72 ℃ are extended 10min again.
1.9 the electrophoresis of PCR product and recovery purifying
The PCR product is identified in 1% agarose gel electrophoresis, determines the size of purpose band with two kinds of nucleic acid molecule amount Marker (DL-2000 and λ-EcoT14 I digest).Concrete grammar is:
1 * TAE (40mmol/L Tris-acetate, 1mmol/L EDTA) damping fluid is made into the sepharose solution of desired concn, microwave oven heating makes after agarose dissolves fully, adding ethidium bromide (5mg/ml EB) to final concentration is 0.5g/ml, pour into behind the mixing in the selected as required good electrophoresis rubber moulding, gel thicknesses is (can be suitably thicker during Rapid identification) between 3～5mm, inserts the suitable comb of width, and comb is apart from the about 0.5～1mm of base plate.Treat gel solidify fully the back (about 30～45min), carefully extract comb, gel is put into the electrophoresis chamber of the electrophoretic buffer that 0.5 * TAE is housed, make electrophoretic buffer just not have glue face (about 1mm).Get then treat in right amount electrophoretic DNA sample drop on the Parafilm film with electrophoresis sample-loading buffer (0.25% tetrabromophenol sulfonphthalein of 1/6 volume, 0.25% dimethylbenzene green grass or young crops, 40% sucrose) mixing is added to biased sample in the loading slot with micro sample adding appliance, with the voltage electrophoresis of 5V/cm.When the tetrabromophenol sulfonphthalein electrophoresis to the appropriate location, observe and the record result with long-wave ultra violet lamp.
With dna fragmentation rapid extraction/recovery test kit (Vitagene company product) the purpose fragment is reclaimed purifying.Concrete operations are:
(1) under ultraviolet lamp, downcuts the sepharose that contains target DNA, exhaust gel surface liquid and chopping with paper handkerchief.Calculated for gel weight (writing down 1.5ml centrifuge tube weight in advance), this weight is as a gel volume (as 100mg=100l);
(2) according to gel strength, add sufficient quantity DE-A solution, mix the back in 75 ℃ of heating (the low melting-point agarose gel is in 45 ℃ of heating), be interrupted and mix, melt (about 6-8 minute) fully until gel piece;
(3) add the DE-B solution of 0.5 DE-A volume, mix; When separated DNA fragment during less than 400bp, adding Virahol to final concentration is 20%;
(4) mixed solution in the absorption (3) is transferred to DNA preparation pipe, the centrifugal 1min of 3600rpm.As prepare liquid residue is arranged in the pipe, suitably improving centrifugal speed, centrifugal again 1min abandons filtrate;
(5) will prepare pipe and put back centrifuge tube, and add 0.5mlW1 solution, the centrifugal 30s of 3600rpm abandons filtrate;
(6) will prepare pipe and put back centrifuge tube, and add 0.7mlW2 solution, the centrifugal 30s of 3600rpm abandons filtrate.Use the 0.7mlW2 solution washing more once with same method;
(7) will prepare pipe and place centrifuge tube, the centrifugal 1min of top speed;
(8) will prepare pipe and place clean 1.5ml centrifuge tube, and prepare the film centre at DNA and add 25l water or elutriant, room temperature leaves standstill 1min.The centrifugal 1min eluted dna of top speed.
2. the structure of recombinant expression plasmid
2.1 the structure of interstitial granules among the fusion gene ORF2-ORF1
Behind restriction enzyme SpeI, SmaI double digestion pMD18T-ORF1 plasmid, reclaim PVC2 ORF1 gene, use SpeI, SmaI double digestion pMD18T-ORF2 plasmid simultaneously after, reclaim the big fragment that contains PCV2 ORF2 gene.The latter is connected with the ORF1 gene is directed, makes up the middle interstitial granules pMD18T-ORF2ORF1 that contains fusion gene.
2.2 contain the structure of interstitial granules in the FMDV P1 gene recombination Fowlpox virus vector
To obtain the P1 gene with the pKSP1 plasmid of Spe I digestion, after flat through mending, the terminal dephosphorization, mend flat, terminal dephosphorization with the pKS linear carrier of ClaI digestion after, both connect and transform the back recombinant plasmid pKSP1 is identified that direction is positioned at the upstream to guarantee HindIII.PKSP1 obtains the P1 gene with HindIII/SalI digestion recombinant plasmid, it is connected with pUTA2L behind the pUTA2-16Lac Z that digests with same enzyme obtain recombinant plasmid pUTA2LP1.
2.3 the structure of coexpression recombinant Borrel virus carrier transferring plasmid
Use HincII and SmaI double digestion plasmid pMD18T-ORF2 and pMD18T-ORF2ORF1 respectively, reclaim ORF2 gene and ORF2ORF1 fusion gene, be connected with the pUTALP1 that cuts also dephosphorization through the SmaI enzyme respectively, make up ORF2 gene and ORF2-ORF1 fusion gene respectively with P1 coexpression recombinant Borrel virus carrier transferring plasmid pUTALP1-ORF2, pUTALP1-ORF2ORF1.
3. the screening of homologous recombination and recombinant virus, purifying
3.1 FPV poison valency is measured
The FPV that is used for homologous recombination calculates plaque forming unit (PFU) through the passage rejuvenation, and dyes identifying virus with HE.FPV is pressed 10
2 ~10
6Extension rate is inoculated in 6 * 30mm culture plate CEF individual layer of the growth of going down to posterity, and add the MEM conduct that contains 1% methylcellulose gum and 1% calf serum (FCS) and keep liquid, 37 ℃, 5%CO
2After cultivating 120h, abandon nutrient solution, PBS (pH7.2) washes 2 times, 1% formaldehyde fixed 15min under the room temperature, the tap water washing, 0.1% violet staining 5min, tap water washing, statistics virus plaque number calculate plaque forming unit contained in every milliliter of viral liquid (Plaque forming units, PFU).
PFU=(virus plaque number * extension rate)/contamination volume (ml)
3.2 homologous recombination in the body
The transferring plasmid transfectional cell adopts liposome method: the CEF 1 * 10 that inoculation is gone down to posterity in 6 * 30mm culture plate
5~ 3 * 10
5Individual/ml, when cell grows to 80% fusion, infect the FPV of 0.1MOI (Multiplicity ofinfection), 37 ℃, 5%CO
2Behind the absorption 2h, with the mixture cotransfection of transfection reagent DOTAP that at room temperature acts on 15 ~ 30min and recombinant plasmid.Promptly in 500 μ l MEM, add 8 μ lLipofectamine
TMReagent, mixing gently, other gets 500 μ l MEM and adds recombinant plasmid dna 10 μ g mixings.Then the latter is dripped in last liquid mixing gently, the greenhouse is effect 15 ~ 30min down.12 ~ 18h after the transfection changes the freshly prepared MEM that contains 2%FCS, continues to cultivate 48 ~ 72h, results virus.The malicious valency of check weighing papova.
3.3 the screening of recombinant virus and purifying
Prepare the CEF individual layer at 6 * 30mm culture plate, connecing malicious preceding 24 hours with the MEM nutritive medium cultivation that contains 40 μ g/ml BUdR (5-bromine ribodesose uridine), then by the virus of gathering in the crops after the transfection of 10MOI inoculation recombinant expression plasmid, behind the 5%CO2 absorption 2h, cultivate 120h, harvested cell with the MEM nutritive medium that contains 40 μ g/ml BUdR again.Repeat above-mentioned experiment, with harvested cell multigelation three times, in the nutritive medium of no BUdR, cultivate then, treat that pathology appears in cell after, choose the single virus plaque respectively, behind the purifying 3 times, expand poison respectively.MEM nutritive medium with no BudR is cultivated, and waits to occur to choose the single virus plaque after the cytopathy, and expands poison respectively.
4. the PCR of recombinant virus identifies
The cell sleaker scrapes cell, the centrifugal 5min of 4000rpm, collecting cell.With PBS (pH7.4) re-suspended cell, thrum repeats twice.4ul 100ug/m Proteinase K is added in the 400ul cell pyrolysis liquid (1%SDS, 10mM NaCl, 10mM EDTA, 10mM Tris-HCl pH8.0), and 56 ℃ of water-bath 24h are bright to lysate.Then with equal-volume phenol/imitative extracting, 12, the centrifugal 10min of 000rpm.Add 800ul dehydrated alcohol and 80ulNH in the supernatant liquor
4AC ,-20 ℃ are spent the night.4 ℃ 12, the centrifugal 10min of 000rpm dries the back and acts on 1 hour, electrophoresis observation for 37 ℃ with an amount of RTE.Get final product from wherein getting 1～5ul during PCR.After the PCR reaction conditions is 95 ℃ of pre-sex change 5min, by 94 ℃ of sex change 60s, 61 ℃ of annealing 60s, 72 ℃ are extended 1min totally 30 circulations, and last 72 ℃ are extended 10min.
5. the detection of expression of recombinant virus product
5.1 indirect immunofluorescence is identified recombinant virus
Get the recombinant virus of an amount of preliminary screening and purifying, be inoculated in CEF monolayer cell on the film flying, establish the contrast of FPV and cell simultaneously.37 ℃, 5%CO
22h is made in sense, adds the MEM nutrient solution that contains 2%FCS, after continuing to cultivate cell and obvious pathology occurring, takes out film flying, and PBS (pH7.2) washes once, 4% Paraformaldehyde 96 fixedly 30min-3 hour, and PBST washes.Use 3% bovine serum albumin damping fluid PBST (1*PBS again, 0.05%Tween20) behind the solution sealing 2h, with pig PCV2 positive serum (1: 300) reaction 2h, PBST washing 3 times, then with the goat-anti pig IgG of fluorescein isothiocyanate (FITC) mark (1: 100-1: 400) reaction 2h, PBST washing 3 ~ 5 times adds a glycerine damping fluid (50% glycerine/PBST) on the slide glass, the film flying that is loaded with cell is inverted on the slide glass, under fluorescent microscope, observes and take pictures.
When the indirect immunofluorescence that carries out FMDV P1 detected, one anti-ly was the anti-FMDV polyclonal antibody of rabbit, and two anti-ly are the goat anti-rabbit igg of fluorescein isothiocyanate (FITC) mark, and all the other reagent and step are the same.
5.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Purification of Recombinant virus with after the amplification is inoculated in 10ml culturing bottle 1 * 10 respectively with 10MOI
6In individual/mlCEF cell, establish FPV contrast and cell simultaneously and contrast, when obvious pathology appears in cell, abandon nutrient solution, with TEN (40mmol/L TrisCl pH7.5, the 1mmol/L EDTA of 37 ℃ of preheatings of 1ml, 150mmol/LNaCl) wash-out cell, be collected in the Eppendorf pipe, the centrifugal 5min of 3000r/min abandons supernatant, cell precipitation is washed once with PBS, add 60 μ l lysis buffer (10mmol/L TrisCl, pH7.4,1mmol/LMgCl
2, 0.5%NP40,20 μ g/ml DNase I) and cracking, ice bath 30min boils 3min, the centrifugal 5min of 5000r/min ,-20 is frozen.
Get 30 μ l cell pyrolysis liquids and equivalent 2 * sample buffer (100mmol/L TrisCl, pH6.8,4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine, adding 2.5% beta-mercaptoethanol before using) mixing, carry out the SDS-PAGE electrophoresis in 10% gel.
5.3 immunoblotting (Western blot)
Behind the SDS-PAGE protein isolate, with its electrotransfer to nitrocellulose filter.5% skimming milk or 3% bovine serum albumin damping fluid (10mmol/L TrisCl, pH7.5,150mmol/L NaCl, 0.05%Tween20) 37 ℃ of sealing 2h, lavation buffer solution (10mmol/L TrisCl pH7.5,150mmol/L NaCl, 0.05%Tween20) washing is 3 times, each 5 ~ 10min, 2 type pig circular ring virus positive serums are with on sealing damping fluid dilution (1: 20) mulch film, 2h is made in the room temperature sense, washs 3 ~ 5 times, and 2h is made in goat-anti pig IgG (1: 300) the room temperature sense of horseradish peroxidase-labeled, after washing 3 ~ 5 times, each 5 ~ 10min is with 10mlDAB damping fluid (dissolving 6mg diaminobenzidine (DAB) in the 10mM of 9ml Tris.HCl (pH7.6) solution, the NiCl of adding 1ml0.3% (w/v)
2/ CoCl
2, to remove the throw out that may form, add 15l 30%H with Whatmanl filter paper filtering substrate solution
2O
2), use immediately behind the mixing.Washed film is moved in the shallow pallet, press 0.1ml/cm
2Add DAB substrate reactions liquid, shake gently in room temperature and carry out the incubation colour developing.Develop the color and move to nitrocellulose filter in the distilled water after 5～20 minutes or use the vitriol oil, color development stopping, observations.
When the Western blot that carries out FMDV P1 detected, one anti-ly was the anti-FMDV polyclonal antibody of rabbit, and two anti-ly are the goat anti-rabbit igg of horseradish peroxidase-labeled, and all the other reagent and step are the same.
6. the stability of expression of recombinant virus foreign protein
With the recombinant virus that obtains, passed for 10 generations continuously with the CEF cell, respectively with the 5th and the 10th generation recombinant virus with the CEF cell of the amount of 10MOI inoculation isodose, the total protein of cell of results is made SDS-PAGE and Western blot analyzes, the proteic relative expression quantity of testing goal.
7. the detected result of expression of recombinant virus product
Behind the recombinant Borrel virus vPUTAL-P1-ORF2 and vPUTAL-ORF2ORF1-P1 inoculated into chick embryo inoblast that screening obtains, locate, all can be observed special yellow-green fluorescence, illustrate that expression product is special at the cytoplasm of cells infected and cytolemma etc.; Analyze through Western blot: the cell culture analysis of reorganization poison inoculation, having occurred to be the special band of 79kD with the size of foot-and-mouth disease virus resistant antibodies, has proved that P1 albumen has obtained expression in chick embryo fibroblast; The cell culture analysis of reorganization poison inoculation, occurred can with the resisting porcine circovirus specific bands (about 25kD, 65kD) of anti-different sizes that combine how.8. the immunogenicity research of recombinant virus
8.1 recombinant virus immune mouse
Recombinant virus inoculation CEF monolayer cell is cultivated 40h, collects cells infected.4 ℃ of 3000r/min are from 10min, cell precipitation (the nutrient solution supernatant is stand-by) is suspended in 10mmol/L TrisCl (pH8.0) solution, in ultrasonic disruption on ice 4 * 15 seconds, with 4 ℃ of centrifugal 10min of 3000r/min of this suspension, get supernatant and measure viral PFU, the titre of adjusting virus makes it reach 107PFU/100ul.
36 of BALB/c female mices, body weight 18～20g divides 3 groups at random, takes injection system under the sufficient lift, injects recombinant Borrel virus vUTAL-P1-ORF2 and vUTAL-P1-ORF2ORF1 respectively and does not contain each 0.1ml of the wild poison of FPV of any foreign gene.The all immunity three times of above-mentioned each group, 20 days at interval, to pluck eyeball in the 10th day after the last immunity to get blood, the cervical vertebra dislocation is put to death, and the blood coagulation separation of serum detects the antigenic IgG antibody of anti-PCV2 for ELISA.Aseptic quantity of getting its spleen with flow cytometry analysis t lymphocyte subset class.
8.2 the detection of spleen amynologic index
8.2.1 spleen list lymphocyte suspension preparation
Mouse is put to death in the cervical vertebra dislocation, and aseptic condition takes out spleen down, places the plate that fills the PBS substratum, grinds with slide, and 200 order nylon net filters are made single cell suspension, 1500r/min, and centrifugal 5min abandons supernatant.With the centrifugal cell twice of washing of Hanks liquid, be resuspended in the RPMI RPMI-1640 that contains 10%NBS, counting transfers to 2 * 10
7Individual/ml is standby.
8.2.2 the detection of spleen t lymphocyte subset class quantity
Extracting spleen cell suspension 0.1ml adds 5ml PBS, 1500r/min, and centrifugal 10min washes cell twice, adds fluorescent mark rat anti-mouse CD in 0.5ml PBS cell suspending liquid respectively
4+And CD
8+Monoclonal antibody (this antibody dilutes by 1: 10 with PBS) 4 warm lucifuges are placed 30min, add 5ml PBS again and wash once, and the centrifugal 10min of 1500r/min will manage floor cells and suspend with 200 μ l PBS, treat that the upflowing cell instrument detects.FACS detects 10000 cells, and the gained data are carried out statistical procedures.
8.2.3 splenocyte Specific CTL Cells cytotoxic activity detects
The preparation of target cell at first makes up plasmid pDISPLAY-P1.Foot and mouth disease virus P1 gene fragment is inserted into the multiple clone site in plasmid pDISPLAY promotor CMV downstream, and construction recombination plasmid pDISPLAY-P1 is used for the preparation of target cell.This plasmid 5g and liposome 8l are softly mixed back transfection P815 cell to be cultivated.
The determining of G418 concentration cultivated 3 days with transfection P815 cell in the RPMI-1640 that contains 10% foetal calf serum after, the dilution G418 of difference is joined the nutrient solution screening of pressurizeing, the growth conditions of observation of cell obtains suitable G418 interpolation concentration.
Change cultivations of going down to posterity of cell after the pressurization screening transfection that the goal gene plasmid cell is arranged, and in its nutrient solution, add the G418 that determines suitable concn, continue cultivation.After the repeated multiple times pressurization screening, can normal growth up to cell, and cell reaches the quantity that is enough to as target cell and irritation cell.If temporarily need not, can in liquid nitrogen, store standby.
The preparation of irritation cell adjusts to 1 * 10 after using the target cell effect 4h of mitomycin (80 μ g/ml) to above-mentioned preparation
6Individual/ml, standby.
Spleen, grinding, filtration are catched and killed, take a blood sample, got to the preparation of splenocyte with mouse, with the washing of Hank ' s liquid, abandons supernatant for the last time, again with the RPMI-1640 (IL-2, the 2 μ g/ml that contain 10% calf serum; Con A, 20ng/ml) the resuspended splenocyte of 1ml, upper plate is cultivated after 1 day for 37 ℃, adds irritation cell effect 24～48h by 10: 1, adjusts spleens cell number to 1 * 10 again
7Individual/ml (note: must with irritation cell and the abundant mixing of splenocyte) continues to cultivate 4-5 days.
CTL is active to be detected
1) effector cell's numeration counts the effector cell in each sample panel, draws cell count at last and reaches 1 * 10
6Individual/ml.
2) numeration of target cell will count through the target cell of G418 pressurization screening, calculates cell quantity.
3) all with after the Hank ' s liquid washing once, the RPMI-1640 of using 5% calf serum for the last time is suspension cell again with two kinds of cells in the processing of splenocyte and target cell.
4) upper plate will be through the effector cell (4 * 10 of above-mentioned processing
6Individual cell/ml) and target cell add respectively in the 96 porocyte growth plate, and each sample is established 8 holes, and wherein 4 holes add target cell, and by the effector cell: target cell was respectively 100: 1 and 50: 1.Experiment test hole and control wells are set, and concrete grammar sees the following form.
5) the CTL killing activity detects
(1) with above-mentioned 37 ℃, 5%CO
2At least after cultivating 4h in the incubator, splenocyte is fully contacted with target cell.
(2) 45min before to be detected adds 10l lysate (10 *) in the target cell hole of containing 100l, target cell serum lactic dehydrogenase in contrast (Lactate dehydrogenase, LDH) calculate by maximum releasing value.(noting:, can add lysate 4l again) if the target cell cracking is incomplete
(3) wait to act on complete back (after being 4h), the centrifugal 4min of 250g.
(4) carefully from each hole of 96 orifice plates, shift out 50l liquid with pipettor, the respective aperture interior (noting the sample order) that adds an other elisa plate respectively, adding the substrate mixture 50l effect 30min that disposes with substrate buffer solution in advance again (notes: place rapidly after substrate and substrate mixture use up in-20 ℃ of refrigerators, must not be positioned over for a long time under 37 ℃ of conditions; In the two mechanism, must use shadings such as platinum paper).
(5) behind the effect 30min, add sulfuric acid stop buffer 50l, on microplate reader, detect behind the thorough mixing.
(6) according to the killing activity that records every group of splenocyte of data computation:
8.3 anti-PCV2 antibody in the ELISA kit measurement immune serum
Envelope antigen in the ELISA test kit is the ORF2 capsid protein of baculovirus expression, and two anti-are the goat anti-mouse igg antibody of horseradish peroxidase-labeled.
In the porous plate of " U " type, the tested serum pref of each part is become 2 parts of 50l, the serum dilution of twice serial dilution, initial titer is 1: 4, adds corresponding one and resist in each hole, hatches 1h at 37 ℃, the extent of dilution of serum rises to 1: 64 always;
With PBS liquid elisa plate is washed 5 times;
Every hole adds two resistive connection compounds of goat-anti mouse immuning ball protein horseradish peroxidase, place the rotation oscillator on 37 ℃ hatch 1h, wash plate;
Every hole of developing the color adds 50l and contains 0.05%H
2O
2Benzidine solution (30%W/V);
Stop with 50l 1.25M sulphuric acid soln termination reaction 15min.
Be determined at enzyme linked immunological instrument 620nm and measure each hole OD value.
The contrast contrast is made up of following: for the antigen of each use, with 4～8 holes, increase serum does not only add the antigen of 50l with PBS (containing 0.05%Tween-20 and phenol red indicator) dilution, corresponding mouse resisting anteserum, 2 times of serial dilutions, each extent of dilution 2 hole; Negative mice serum, 2 times of serial dilutions, each extent of dilution 2 hole.
The result judges that antibody titers can reach with 50% final extent of dilution of the virus control hole photoabsorption mean value of no tested serum with tested serum and represents.Titre is positive above 1: 40, and titre should use virus neutralization experiment heavily to examine near 1: 40 o'clock.
9. recombinant virus is in the immunology research of body animal pig
Immunology and detection method with mouse.
Detected result:
Two strain recombinant Borrel virus vPUTAL-ORF2-P1 that obtain and vPUTAL-ORF2ORF1-P1 extract the laggard performing PCR amplification of genome, all must expect the purpose fragment of size.Indirect immunofluorescence assay and Westernblot detect and are positive findings, illustrate that the recombinant Borrel virus of structure can be expressed foot and mouth disease virus structural protein precursor P1 and pig circular ring virus 2 virus capsid protein and replication protein respectively.Two strain recombinant Borrel virus all can be expressed in non-poultry animal cell, and recombinant virus has good genetic stability.With carrying out the mouse immune experiment after a large amount of amplifications of recombinant Borrel virus, can in immune serum, detect the antibody of anti-FMDV structural protein and PCV2 capsid protein, show that recombinant virus stimulates the mouse body to produce specific humoral immunity.The detection and the CTL fragmentation test result of immune mouse splenic t-cell subclass quantity show that all mouse has produced specific cellular immunity.Recombinant Borrel virus is carried out the pig immunization experiment, obtained same conclusion.
Result of study shows that two strain recombinant Borrel virus are a kind of safe and effective genetically engineered live vector vaccines, and having exploitation becomes the good prospect that is used for the preventative and therapeutic vaccine of pmws and foot and mouth disease.
<210>1
<211>702
<212>DNA
<213>virus
＜220〉2 type Chinese epidemic strain PCV-ORF2 sequences
<221>gene
<222>(1)..(702)
<400>1
atg?acg?tat?cca?agg?agg?cgt?tac?cgg?aga?aga?aga?cac?cgc?ccc?cgc 48
Met?Thr?Tyr?Pro?Arg?Arg?Arg?Tyr?Arg?Arg?Arg?Arg?His?Arg?Pro?Arg
1 5 10 15
agc?cat?ctt?ggc?cag?atc?ctc?cgc?cgc?cgc?ccc?tgg?ctc?gtc?cac?ccc 96
Ser?His?Leu?Gly?Gln?Ile?Leu?Arg?Arg?Arg?Pro?Trp?Leu?Val?His?Pro
20 25 30
cgc?cac?cgt?tac?cgc?tgg?aga?agg?aaa?aat?ggc?atc?ttc?aac?acc?cgc 144
Arg?His?Arg?Tyr?Arg?Trp?Arg?Arg?Lys?Asn?Gly?Ile?Phe?Asn?Thr?Arg
35 40 45
ctc?tcc?cgc?acc?ttc?gga?tat?act?atc?aag?cga?acc?aca?gtc?aaa?acg 192
Leu?Ser?Arg?Thr?Phe?Gly?Tyr?Thr?Ile?Lys?Arg?Thr?Thr?Val?Lys?Thr
50 55 60
ccc?tcc?tgg?gcg?gtg?gac?atg?atg?aca?ttc?aat?att?aat?gac?ttt?ctt 240
Pro?Ser?Trp?Ala?Val?Asp?Met?Met?Thr?Phe?Asn?Ile?Asn?Asp?Phe?Leu
65 70 75 80
ccc?cca?gga?ggg?ggc?tca?aac?ccc?cgc?tct?gtg?ccc?ttt?gaa?tac?tac 288
Pro?Pro?Gly?Gly?Gly?Ser?Asn?Pro?Arg?Ser?Val?Pro?Phe?Glu?Tyr?Tyr
85 90 95
aga?ata?aga?aag?gtt?aag?gtt?gaa?ttc?tgg?ccc?tgc?tcc?ccg?atc?acc 336
Arg?Ile?Arg?Lys?Val?Lys?Val?Glu?Phe?Trp?Pro?Cys?Ser?Pro?Ile?Thr
100 105 110
cag?ggt?gac?agg?gga?gtg?ggc?tcc?agt?gct?gtt?att?cta?gat?gat?aac 384
Gln?Gly?Asp?Arg?Gly?Val?Gly?Ser?Ser?Ala?Val?Ile?Leu?Asp?Asp?Asn
115 120 125
ttt?gta?aca?aag?gcc?aca?gcc?ctc?acc?tat?gac?ccc?tat?gta?aac?tac 432
Phe?Val?Thr?Lys?Ala?Thr?Ala?Leu?Thr?Tyr?Asp?Pro?Tyr?Val?Asn?Tyr
130 135 140
tcc?tcc?cgc?cat?acc?ata?acc?cag?ccc?ttc?tcc?tac?cac?tcc?cgc?tac 480
Ser?Ser?Arg?His?Thr?Ile?Thr?Gln?Pro?Phe?Ser?Tyr?His?Ser?Arg?Tyr
145 150 155 160
ttt?acc?ccc?aaa?cct?gtc?cta?gat?tcc?act?att?gat?tac?ttc?caa?cca 528
Phe?Thr?Pro?Lys?Pro?Val?Leu?Asp?Ser?Thr?Ile?Asp?Tyr?Phe?Gln?Pro
165 170 175
aac?aac?aaa?aga?aat?cag?ctg?tgg?ctg?aga?cta?caa?act?gct?gga?aat 576
Asn?Asn?Lys?Arg?Asn?Gln?Leu?Trp?Leu?Arg?Leu?Gln?Thr?Ala?Gly?Asn
180 185 190
gta?gac?cac?gta?ggc?ctc?ggc?act?gcg?ttc?gaa?aac?agt?ata?tac?gac 624
Val?Asp?His?Val?Gly?Leu?Gly?Thr?Ala?Phe?Glu?Ash?Ser?Ile?Tyr?Asp
195 200 205
cag?gaa?tac?aat?atc?cgt?gta?acc?atg?tat?gta?caa?ttc?aga?gaa?ttt 672
Gln?Glu?Tyr?Asn?Ile?Arg?Val?Thr?Met?Tyr?Val?Gln?Phe?Arg?Glu?Phe
210 215 220
aat?ctt?aaa?gac?ccc?cca?ctt?aac?ccc?taa 702
Asn?Leu?Lys?Asp?Pro?Pro?Leu?Asn?Pro
225 230
<210>2
<211>1734
<212>DNA
<213>virus
＜220〉2 type Chinese epidemic strain PCV-ORF1+ORF2 sequences
<221>gene
<222>(1)..(1734)
<400>2
atg?acg?tat?cca?agg?agg?cgt?tac?cgg?aga?aga?aga?cac?cgc?ccc?cgc 48
Met?Thr?Tyr?Pro?Arg?Arg?Arg?Tyr?Arg?Arg?Arg?Arg?His?Arg?Pro?Arg
1 5 10 15
agc?cat?ctt?ggc?cag?atc?ctc?cgc?cgc?cgc?ccc?tgg?ctc?gtc?cac?ccc 96
Ser?His?Leu?Gly?Gln?Ile?Leu?Arg?Arg?Arg?Pro?Trp?Leu?Val?His?Pro
20 25 30
cgc?cac?cgt?tac?cgc?tgg?aga?agg?aaa?aat?ggc?atc?ttc?aac?acc?cgc 144
Arg?His?Arg?Tyr?Arg?Trp?Arg?Arg?Lys?Asn?Gly?Ile?Phe?Asn?Thr?Arg
35 40 45
ctc?tcc?cgc?acc?ttc?gga?tat?act?atc?aag?cga?acc?aca?gtc?aaa?acg 192
Leu?Ser?Arg?Thr?Phe?Gly?Tyr?Thr?Ile?Lys?Arg?Thr?Thr?Val?Lys?Thr
50 55 60
ccc?tcc?tgg?gcg?gtg?gac?atg?atg?aca?ttc?aat?att?aat?gac?ttt?ctt 240
Pro?Ser?Trp?Ala?Val?Asp?Met?Met?Thr?Phe?Asn?Ile?Asn?Asp?Phe?Leu
65 70 75 80
ccc?cca?gga?ggg?ggc?tca?aac?ccc?cgc?tct?gtg?ccc?ttt?gaa?tac?tac 288
Pro?Pro?Gly?Gly?Gly?Ser?Asn?Pro?Arg?Ser?Val?Pro?Phe?Glu?Tyr?Tyr
85 90 95
aga?ata?aga?aag?gtt?aag?gtt?gaa?ttc?tgg?ccc?tgc?tcc?ccg?atc?acc 336
Arg?Ile?Arg?Lys?Val?Lys?Val?Glu?Phe?Trp?Pro?Cys?Ser?Pro?Ile?Thr
100 105 110
cag?ggt?gac?agg?gga?gtg?ggc?tcc?agt?gct?gtt?att?cta?gat?gat?aac 384
Gln?Gly?Asp?Arg?Gly?Val?Gly?Ser?Ser?Ala?Val?Ile?Leu?Asp?Asp?Asn
115 120 125
ttt?gta?aca?aag?gcc?aca?gcc?ctc?acc?tat?gac?ccc?tat?gta?aac?tac 432
Phe?Val?Thr?Lys?Ala?Thr?Ala?Leu?Thr?Tyr?Asp?Pro?Tyr?Val?Asn?Tyr
130 135 140
tcc?tcc?cgc?cat?acc?ata?acc?cag?ccc?ttc?tcc?tac?cac?tcc?cgc?tac 480
Ser?Ser?Arg?His?Thr?Ile?Thr?Gln?Pro?Phe?Ser?Tyr?His?Ser?Arg?Tyr
145 150 155 160
ttt?acc?ccc?aaa?cct?gtc?cta?gat?tcc?act?att?gat?tac?ttc?caa?cca 528
Phe?Thr?Pro?Lys?Pro?Val?Leu?Asp?Ser?Thr?Ile?Asp?Tyr?Phe?Gln?Pro
165 170 175
aac?aac?aaa?aga?aat?cag?ctg?tgg?ctg?aga?cta?caa?act?gct?gga?aat 576
Asn?Asn?Lys?Arg?Asn?Gln?Leu?Trp?Leu?Arg?Leu?Gln?Thr?Ala?Gly?Asn
180 185 190
gta?gac?cac?gta?ggc?ctc?ggc?act?gcg?ttc?gaa?aac?agt?ata?tac?gac 624
Val?Asp?His?Val?Gly?Leu?Gly?Thr?Ala?Phe?Glu?Asn?Ser?Ile?Tyr?Asp
195 200 205
cag?gaa?tac?aat?atc?cgt?gta?acc?atg?tat?gta?caa?ttc?aga?gaa?ttt 672
Gln?Glu?Tyr?Asn?Ile?Arg?Val?Thr?Met?Tyr?Val?Gln?Phe?Arg?Glu?Phe
210 215 220
aat?ctt?aaa?gac?ccc?cca?ctt?aac?cct?tat?act?agt?ctg?aaa?acg?aaa 720
Asn?Leu?Lys?Asp?Pro?Pro?Leu?Asn?Pro?Tyr?Thr?Ser?Leu?Lys?Thr?Lys
225 230 235 240
gaa?gtg?cgc?tgt?aag?tat?tac?cag?cgc?act?tcg?gca?gcg?gca?gca?cct 768
Glu?Val?Arg?Cys?Lys?Tyr?Tyr?Gln?Arg?Thr?Ser?Ala?Ala?Ala?Ala?Pro
245 250 255
cgg?cag?cac?ctc?agc?agc?aac?atg?ccc?agc?aag?aag?aat?gga?aga?agc 816
Arg?Gln?His?Leu?Ser?Ser?Asn?Met?Pro?Ser?Lys?Lys?Asn?Gly?Arg?Ser
260 265 270
gga?ccc?caa?ccc?cat?aaa?agg?tgg?gtg?ttc?act?ctg?aat?aat?cct?tcc 864
Gly?Pro?Gln?Pro?His?Lys?Arg?Trp?Val?Phe?Thr?Leu?Asn?Asn?Pro?Ser
275 280 285
gaa?gac?gag?cgc?aag?aaa?ata?cgg?gat?ctt?cca?ata?tcc?cta?ttt?gat 912
Glu?Asp?Glu?Arg?Lys?Lys?Ile?Arg?Asp?Leu?Pro?Ile?Ser?Leu?Phe?Asp
290 295 300
tat?ttt?att?gtt?ggc?gag?gag?ggt?aat?gag?gaa?gga?cga?aca?cct?cac 960
Tyr?Phe?Ile?Val?Gly?Glu?Glu?Gly?Asn?Glu?Glu?Gly?Arg?Thr?Pro?His
305 310 315 320
ctc?cag?ggg?ttc?gct?aat?ttt?gtg?aag?aag?cag?act?ttt?aat?aaa?gtg 1008
Leu?Gln?Gly?Phe?Ala?Asn?Phe?Val?Lys?Lys?Gln?Thr?Phe?Asn?Lys?Val
325 330 335
aag?tgg?tat?ttg?ggt?gcc?cgc?tgc?cac?atc?gag?aaa?gcg?aaa?gga?aca 1056
Lys?Trp?Tyr?Leu?Gly?Ala?Arg?Cys?His?Ile?Glu?Lys?Ala?Lys?Gly?Thr
340 345 350
gat?cag?cag?aat?aaa?gaa?tac?tgc?agt?aaa?gaa?ggc?aac?tta?ctg?atg 1104
Asp?Gln?Gln?Asn?Lys?Glu?Tyr?Cys?Ser?Lys?Glu?Gly?Asn?Leu?Leu?Met
355 360 365
gag?tgt?gga?gct?cct?aga?tct?cag?gga?caa?cgg?agt?gac?ctg?tct?act 1152
Glu?Cys?Gly?Ala?Pro?Arg?Ser?Gln?Gly?Gln?Arg?Ser?Asp?Leu?Ser?Thr
370 375 380
gct?gtg?agt?acc?ttg?ttg?gag?agc?ggg?agt?ctg?gtg?acc?gtt?gca?gag 1200
Ala?Val?Ser?Thr?Leu?Leu?Glu?Ser?Gly?Ser?Leu?Val?Thr?Val?Ala?Glu
385 390 395 400
cag?cac?cct?gta?acg?ttt?gtc?aga?aat?ttc?cgc?ggg?ctg?gct?gaa?ctt 1248
Gln?His?Pro?Val?Thr?Phe?Val?Arg?Asn?Phe?Arg?Gly?Leu?Ala?Glu?Leu
405 410 415
ttg?aaa?gtg?agc?ggg?aaa?atg?cag?aag?cgt?gat?tgg?aag?act?aat?gta 1296
Leu?Lys?Val?Ser?Gly?Lys?Met?Gln?Lys?Arg?Asp?Trp?Lys?Thr?Asn?Val
420 425 430
cac?gtc?att?gtg?ggg?cca?cct?ggg?tgt?ggt?aaa?agc?aaa?tgg?gct?gct 1344
His?Val?Ile?Val?Gly?Pro?Pro?Gly?Cys?Gly?Lys?Ser?Lys?Trp?Ala?Ala
435 440 445
aat?ttt?gca?gac?ccg?gaa?acc?aca?tac?tgg?aaa?cca?cct?aga?aac?aag 1392
Asn?Phe?Ala?Asp?Pro?Glu?Thr?Thr?Tyr?Trp?Lys?Pro?Pro?Arg?Asn?Lys
450 455 460
tgg?tgg?gat?ggt?tac?cat?ggt?gaa?gaa?gtg?gtt?gtt?att?gat?gac?ttt 1440
Trp?Trp?Asp?Gly?Tyr?His?Gly?Glu?Glu?Val?Val?Val?Ile?Asp?Asp?Phe
465 470 475 480
tat?ggc?tgg?ctg?ccc?tgg?gat?gat?cta?ctg?aga?ctg?tgt?gat?cga?tat 1488
Tyr?Gly?Trp?Leu?Pro?Trp?Asp?Asp?Leu?Leu?Arg?Leu?Cys?Asp?Arg?Tyr
485 490 495
cca?ttg?act?gta?gag?act?aaa?ggt?gga?act?gta?cct?ttt?ttg?gcc?cgc 1536
Pro?Leu?Thr?Val?Glu?Thr?Lys?Gly?Gly?Thr?Val?Pro?Phe?Leu?Ala?Arg
500 505 510
agt?att?ctg?att?acc?agc?aat?cag?acc?ccg?ttg?gaa?tgg?tac?tcc?tca 1584
Ser?Ile?Leu?Ile?Thr?Ser?Asn?Gln?Thr?Pro?Leu?Glu?Trp?Tyr?Ser?Ser
515 520 525
act?gct?gtc?cca?gct?gta?gaa?gct?ctt?tat?cgg?agg?att?act?tcc?ttg 1632
Thr?Ala?Val?Pro?Ala?Val?Glu?Ala?Leu?Tyr?Arg?Arg?Ile?Thr?Ser?Leu
530 535 540
gta?ttt?tgg?aag?aat?gct?aca?gaa?caa?tcc?acg?gag?gaa?ggg?ggc?cag 1680
Val?Phe?Trp?Lys?Asn?Ala?Thr?Glu?Gln?Ser?Thr?Glu?Glu?Gly?Gly?Gln
545 550 555 560
ttc?gtc?acc?ctt?tcc?ccc?cca?tgc?cct?gaa?ttt?cca?tat?gaa?ata?aat 1728
Phe?Val?Thr?Leu?Ser?Pro?Pro?Cys?Pro?Glu?Phe?Pro?Tyr?Glu?Ile?Asn
565 570 575
tac?tga 1734
Tyr
Claims (6)
1, the bigeminy live recombinant vectors vaccine of a kind of coexpression pig circular ring virus and foot and mouth disease virus is characterized in that:
Set up a new pig circular ring virus genomic library: the application PCR method increases the full gene segmentation of Chinese PCR2 epidemic strain nm2002 and is cloned in pMD18-T and the pGEM-T Easy carrier, two plasmids of pMD18T-ORF2 and pGEMT-XS have been made up, splicing and obtain the whole genome sequence of Chinese PCV2 epidemic strain nm2002;
Again gene involved in immunity is cloned into the expression vector from the Central Asia, DNA library that makes up, in host cell, gives expression to corresponding gene, as prevention or to examine protein, genetic recombinants or the recombinant immune of pmws former.
2, the bigeminy live recombinant vectors vaccine of coexpression pig circular ring virus according to claim 1 and foot and mouth disease virus, it is characterized in that: the P1 district gene of FMDV O-NYOO strain is inserted into the sub-downstream of bird pox virus expression vector pUTAL P7.5 tandem promoter from the pKS-P1 carrier after with HindIII and SalI double digestion, form intermediate carrier pUTALP1, after simultaneously the PCV2ORF2 gene of Chinese epidemic strain PCV2nm2002 being used HincII and SmaI double digestion from carrier pMD18T-ORF2, be inserted into bird pox virus expression vector pUTALP1ATI-P7.5 combined promoter downstream, made up coexpression PCV2ORF2 and FMDV O-NYOOP1 recombinant Borrel transferring plasmid pUTALP1-ORF2.
3, the bigeminy live recombinant vectors vaccine of coexpression pig circular ring virus according to claim 2 and foot and mouth disease virus, its gene order is:
atg?acg?tat?cca?agg?agg?cgt?tac?cgg?aga?aga?aga?cac?cgc?ccc?cgc?48
Met?Thr?Tyr?Pro?Arg?Arg?Arg?Tyr?Arg?Arg?Arg?Arg?His?Arg?Pro?Arg
1 5 10 15
agc?cat?ctt?ggc?cag?atc?ctc?cgc?cgc?cgc?ccc?tgg?ctc?gtc?cac?ccc 96
Ser?His?Leu?Gly?Gln?Ile?Leu?Arg?Arg?Arg?Pro?Trp?Leu?Val?His?Pro
20 25 30
cgc?cac?cgt?tac?cgc?tgg?aga?agg?aaa?aat?ggc?atc?ttc?aac?acc?cgc 144
Arg?His?Arg?Tyr?Arg?Trp?Arg?Arg?Lys?Asn?Gly?Ile?Phe?Asn?Thr?Arg
35 40 45
ctc?tcc?cgc?acc?ttc?gga?tat?act?atc?aag?cga?acc?aca?gtc?aaa?acg 192
Leu?Ser?Arg?Thr?Phe?Gly?Tyr?Thr?Ile?Lys?Arg?Thr?Thr?Val?Lys?Thr
50 55 60
ccc?tcc?tgg?gcg?gtg?gac?atg?atg?aca?ttc?aat?att?aat?gac?ttt?ctt 240
Pro?Ser?Trp?Ala?Val?Asp?Met?Met?Thr?Phe?Asn?Ile?Asn?Asp?Phe?Leu
65 70 75 80
ccc?cca?gga?ggg?ggc?tca?aac?ccc?cgc?tct?gtg?ccc?ttt?gaa?tac?tac 288
Pro?Pro?Gly?Gly?Gly?Ser?Asn?Pro?Arg?Ser?Val?Pro?Phe?Glu?Tyr?Tyr
85 90 95
aga?ata?aga?aag?gtt?aag?gtt?gaa?ttc?tgg?ccc?tgc?tcc?ccg?atc?acc 336
Arg?Ile?Arg?Lys?Val?Lys?Val?Glu?Phe?Trp?Pro?Cys?Ser?Pro?Ile?Thr
100 105 110
cag?ggt?gac?agg?gga?gtg?ggc?tcc?agt?gct?gtt?att?cta?gat?gat?aac 384
Gln?Gly?Asp?Arg?Gly?Val?Gly?Ser?Ser?Ala?Val?Ile?Leu?Asp?Asp?Asn
115 120 125
ttt?gta?aca?aag?gcc?aca?gcc?ctc?acc?tat?gac?ccc?tat?gta?aac?tac 432
Phe?Val?Thr?Lys?Ala?Thr?Ala?Leu?Thr?Tyr?Asp?Pro?Tyr?Val?Asn?Tyr
130 135 140
tcc?tcc?cgc?cat?acc?ata?acc?cag?ccc?ttc?tcc?tac?cac?tcc?cgc?tac 480
Ser?Ser?Arg?His?Thr?Ile?Thr?Gln?Pro?Phe?Ser?Tyr?His?Ser?Arg?Tyr
145 150 155 160
ttt?acc?ccc?aaa?cct?gtc?cta?gat?tcc?act?att?gat?tac?ttc?caa?cca 528
Phe?Thr?Pro?Lys?Pro?Val?Leu?Asp?Ser?Thr?Ile?Asp?Tyr?Phe?Gln?Pro
165 170 175
aac?aac?aaa?aga?aat?cag?ctg?tgg?ctg?aga?cta?caa?act?gct?gga?aat 576
Asn?Asn?Lys?Arg?Asn?Gln?Leu?Trp?Leu?Arg?Leu?Gln?Thr?Ala?Gly?Asn
180 185 190
gta?gac?cac?gta?ggc?ctc?ggc?act?gcg?ttc?gaa?aac?agt?ata?tac?gac 624
Val?Asp?His?Val?Gly?Leu?Gly?Thr?Ala?Phe?Glu?Asn?Ser?Ile?Tyr?Asp
195 200 205
cag?gaa?tac?aat?atc?cgt?gta?acc?atg?tat?gta?caa?ttc?aga?gaa?ttt 672
Gln?Glu?Tyr?Asn?Ile?Arg?Val?Thr?Met?Tyr?Val?Gln?Phe?Arg?Glu?Phe
210 215 220
aat?ctt?aaa?gac?ccc?cca?ctt?aac?ccc?taa 702
Asn?Leu?Lys?Asp?Pro?Pro?Leu?Asn?Pro
225 230
4, the bigeminy live recombinant vectors vaccine of coexpression pig circular ring virus according to claim 1 and foot and mouth disease virus, it is characterized in that: the P1 district gene of FMDV O-NYOO strain is inserted into the sub-downstream of bird pox virus expression vector pUTAL P7.5 tandem promoter from the pKS-P1 carrier after with HindIII and SalI double digestion, form intermediate carrier pUTALP1, simultaneously with the middle interstitial granules pMD18T-ORF2-ORF1 of the ORF2-ORF1 fusion gene of Chinese epidemic strain PCV2 nm2002 with HincII and SmaI double digestion after, be inserted into bird pox virus expression vector pUTAL ATI-P7.5 combined promoter downstream, make up coexpression PCV2ORF2-ORF1 and FMDV O-NYOO P1 recombinant Borrel transferring plasmid pUTALP1-ORF2-ORF1.
5, the bigeminy live recombinant vectors vaccine of coexpression pig circular ring virus according to claim 4 and foot and mouth disease virus, its gene order is:
atg?acg?tat?cca?agg?agg?cgt?tac?cgg?aga?aga?aga?cac?cgc?ccc?cgc 48
Met?Thr?Tyr?Pro?Arg?Arg?Arg?Tyr?Arg?Arg?Arg?Arg?His?Arg?Pro?Arg
1 5 10 15
agc?cat?ctt?ggc?cag?atc?ctc?cgc?cgc?cgc?ccc?tgg?ctc?gtc?cac?ccc 96
Ser?His?Leu?Gly?Gln?Ile?Leu?Arg?Arg?Arg?Pro?Trp?Leu?Val?His?Pro
20 25 30
cgc?cac?cgt?tac?cgc?tgg?aga?agg?aaa?aat?ggc?atc?ttc?aac?acc?cgc 144
Arg?His?Arg?Tyr?Arg?Trp?Arg?Arg?Lys?Asn?Gly?Ile?Phe?Asn?Thr?Arg
35 40 45
ctc?tcc?cgc?acc?ttc?gga?tat?act?atc?aag?cga?acc?aca?gtc?aaa?acg 192
Leu?Ser?Arg?Thr?Phe?Gly?Tyr?Thr?Ile?Lys?Arg?Thr?Thr?Val?Lys?Thr
50 55 60
ccc?tcc?tgg?gcg?gtg?gac?atg?atg?aca?ttc?aat?att?aat?gac?ttt?ctt 240
Pro?Ser?Trp?Ala?Val?Asp?Met?Met?Thr?Phe?Asn?Ile?Asn?Asp?Phe?Leu
65 70 75 80
ccc?cca?gga?ggg?ggc?tca?aac?ccc?cgc?tct?gtg?ccc?ttt?gaa?tac?tac 288
Pro?Pro?Gly?Gly?Gly?Ser?Asn?Pro?Arg?Ser?Val?Pro?Phe?Glu?Tyr?Tyr
85 90 95
aga?ata?aga?aag?gtt?aag?gtt?gaa?ttc?tgg?ccc?tgc?tcc?ccg?atc?acc 336
Arg?Ile?Arg?Lys?Val?Lys?Val?Glu?Phe?Trp?Pro?Cys?Ser?Pro?Ile?Thr
100 105 110
cag?ggt?gac?agg?gga?gtg?ggc?tcc?agt?gct?gtt?att?cta?gat?gat?aac 384
Gln?Gly?Asp?Arg?Gly?Val?Gly?Ser?Ser?Ala?Val?Ile?Leu?Asp?Asp?Asn
115 120 125
ttt?gta?aca?aag?gcc?aca?gcc?ctc?acc?tat?gac?ccc?tat?gta?aac?tac 432
Phe?Val?Thr?Lys?Ala?Thr?Ala?Leu?Thr?Tyr?Asp?Pro?Tyr?Val?Asn?Tyr
130 135 140
tcc?tcc?cgc?cat?acc?ata?acc?cag?ccc?ttc?tcc?tac?cac?tcc?cgc?tac 480
Ser?Ser?Arg?His?Thr?Ile?Thr?Gln?Pro?Phe?Ser?Tyr?His?Ser?Arg?Tyr
145 150 155 160
ttt?acc?ccc?aaa?cct?gtc?cta?gat?tcc?act?att?gat?tac?ttc?caa?cca 528
Phe?Thr?Pro?Lys?Pro?Val?Leu?Asp?Ser?Thr?Ile?Asp?Tyr?Phe?Gln?Pro
165 170 175
aac?aac?aaa?aga?aat?cag?ctg?tgg?ctg?aga?cta?caa?act?gct?gga?aat 576
Asn?Asn?Lys?Arg?Asn?Gln?Leu?Trp?Leu?Arg?Leu?Gln?Thr?Ala?Gly?Asn
180 185 190
gta?gac?cac?gta?ggc?ctc?ggc?act?gcg?ttc?gaa?aac?agt?ata?tac?gac 624
Val?Asp?His?Val?Gly?Leu?Gly?Thr?Ala?Phe?Glu?Asn?Ser?Ile?Tyr?Asp
195 200 205
cag?gaa?tac?aat?atc?cgt?gta?acc?atg?tat?gta?caa?ttc?aga?gaa?ttt 672
Gln?Glu?Tyr?Asn?Ile?Arg?Val?Thr?Met?Tyr?Val?Gln?Phe?Arg?Glu?Phe
210 215 220
aat?ctt?aaa?gac?ccc?cca?ctt?aac?cct?tat?act?agt?ctg?aaa?acg?aaa 720
Asn?Leu?Lys?Asp?Pro?Pro?Leu?Asn?Pro?Tyr?Thr?Ser?Leu?Lys?Thr?Lys
225 230 235 240
gaa?gtg?cgc?tgt?aag?tat?tac?cag?cgc?act?tcg?gca?gcg?gca?gca?cct 768
Glu?Val?Arg?Cys?Lys?Tyr?Tyr?Gln?Arg?Thr?Ser?Ala?Ala?Ala?Ala?Pro
245 250 255
cgg?cag?cac?ctc?agc?agc?aac?atg?ccc?agc?aag?aag?aat?gga?aga?agc 816
Arg?Gln?His?Leu?Ser?Ser?Asn?Met?Pro?Ser?Lys?Lys?Asn?Gly?Arg?Ser
260 265 270
gga?ccc?caa?ccc?cat?aaa?agg?tgg?gtg?ttc?act?ctg?aat?aat?cct?tcc 864
Gly?Pro?Gln?Pro?His?Lys?Arg?Trp?Val?Phe?Thr?Leu?Asn?Asn?Pro?Ser
275 280 285
gaa?gac?gag?cgc?aag?aaa?ata?cgg?gat?ctt?cca?ata?tcc?cta?ttt?gat 912
Glu?Asp?Glu?Arg?Lys?Lys?Ile?Arg?Asp?Leu?Pro?Ile?Ser?Leu?Phe?Asp
290 295 300
tat?ttt?att?gtt?ggc?gag?gag?ggt?aat?gag?gaa?gga?cga?aca?cct?cac 960
Tyr?Phe?Ile?Val?Gly?Glu?Glu?Gly?Asn?Glu?Glu?Gly?Arg?Thr?Pro?His
305 310 315 320
ctc?cag?ggg?ttc?gct?aat?ttt?gtg?aag?aag?cag?act?ttt?aat?aaa?gtg 1008
Leu?Gln?Gly?Phe?Ala?Asn?Phe?Val?Lys?Lys?Gln?Thr?Phe?Asn?Lys?Val
325 330 335
aag?tgg?tat?ttg?ggt?gcc?cgc?tgc?cac?atc?gag?aaa?gcg?aaa?gga?aca 1056
Lys?Trp?Tyr?Leu?Gly?Ala?Arg?Cys?His?Ile?Glu?Lys?Ala?Lys?Gly?Thr
340 345 350
gat?cag?cag?aat?aaa?gaa?tac?tgc?agt?aaa?gaa?ggc?aac?tta?ctg?atg 1104
Asp?Gln?Gln?Asn?Lys?Glu?Tyr?Cys?Ser?Lys?Glu?Gly?Asn?Leu?Leu?Met
355 360 365
gag?tgt?gga?gct?cct?aga?tct?cag?gga?caa?cgg?agt?gac?ctg?tct?act 1152
Glu?Cys?Gly?Ala?Pro?Arg?Ser?Gln?Gly?Gln?Arg?Ser?Asp?Leu?Ser?Thr
370 375 380
gct?gtg?agt?acc?ttg?ttg?gag?agc?ggg?agt?ctg?gtg?acc?gtt?gca?gag 1200
Ala?Val?Ser?Thr?Leu?Leu?Glu?Ser?Gly?Ser?Leu?Val?Thr?Val?Ala?Glu
385 390 395 400
cag?cac?cct?gta?acg?ttt?gtc?aga?aat?ttc?cgc?ggg?ctg?gct?gaa?ctt 1248
Gln?His?Pro?Val?Thr?Phe?Val?Arg?Asn?Phe?Arg?Gly?Leu?Ala?Glu?Leu
405 410 415
ttg?aaa?gtg?agc?ggg?aaa?atg?cag?aag?cgt?gat?tgg?aag?act?aat?gta 1296
Leu?Lys?Val?Ser?Gly?Lys?Met?Gln?Lys?Arg?Asp?Trp?Lys?Thr?Asn?Val
420 425 430
cac?gtc?att?gtg?ggg?cca?cct?ggg?tgt?ggt?aaa?agc?aaa?tgg?gct?gct 1344
His?Val?Ile?Val?Gly?Pro?Pro?Gly?Cys?Gly?Lys?Ser?Lys?Trp?Ala?Ala
435 440 445
aat?ttt?gca?gac?ccg?gaa?acc?aca?tac?tgg?aaa?cca?cct?aga?aac?aag 1392
Asn?Phe?Ala?Asp?Pro?Glu?Thr?Thr?Tyr?Trp?Lys?Pro?Pro?Arg?Asn?Lys
450 455 460
tgg?tgg?gat?ggt?tac?cat?ggt?gaa?gaa?gtg?gtt?gtt?att?gat?gac?ttt 1440
Trp?Trp?Asp?Gly?Tyr?His?Gly?Glu?Glu?Val?Val?Val?Ile?Asp?Asp?Phe
465 470 475 480
tat?ggc?tgg?ctg?ccc?tgg?gat?gat?cta?ctg?aga?ctg?tgt?gat?cga?tat 1488
Tyr?Gly?Trp?Leu?Pro?Trp?Asp?Asp?Leu?Leu?Arg?Leu?Cys?Asp?Arg?Tyr
485 490 495
cca?ttg?act?gta?gag?act?aaa?ggt?gga?act?gta?cct?ttt?ttg?gcc?cgc 1536
Pro?Leu?Thr?Val?Glu?Thr?Lys?Gly?Gly?Thr?Val?Pro?Phe?Leu?Ala?Arg
500 505 510
agt?att?ctg?att?acc?agc?aat?cag?acc?ccg?ttg?gaa?tgg?tac?tcc?tca 1584
Ser?Ile?Leu?Ile?Thr?Ser?Asn?Gln?Thr?Pro?Leu?Glu?Trp?Tyr?Ser?Ser
515 520 525
act?gct?gtc?cca?gct?gta?gaa?gct?ctt?tat?cgg?agg?att?act?tcc?ttg 1632
Thr?Ala?Val?Pro?Ala?Val?Glu?Ala?Leu?Tyr?Arg?Arg?Ile?Thr?Ser?Leu
530 535 540
gta?ttt?tgg?aag?aat?gct?aca?gaa?caa?tcc?acg?gag?gaa?ggg?ggc?cag 1680
Val?Phe?Trp?Lys?Asn?Ala?Thr?Glu?Gln?Ser?Thr?Glu?Glu?Gly?Gly?Gln
545 550 555 560
ttc?gtc?acc?ctt?tcc?ccc?cca?tgc?cct?gaa?ttt?cca?tat?gaa?ata?aat 1728
Phe?Val?Thr?Leu?Ser?Pro?Pro?Cys?Pro?Glu?Phe?Pro?Tyr?Glu?Ile?Asn
565 570 575
tac?tga 1734
Tyr
6, the bigeminy live recombinant vectors vaccine of coexpression pig circular ring virus according to claim 1 and foot and mouth disease virus, it is characterized in that: associated protein can obtain expressing in the plant rhabdovirus expression vector from insect cell, also can be animal virus expression vector or the expression of other carrier for expression of eukaryon in mammalian cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410011047 CN1749397B (en) | 2004-08-18 | 2004-08-18 | Bi-recombinant active carrier vaccine of co-expression pig gyrate virus and foot and mouth disease virus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410011047 CN1749397B (en) | 2004-08-18 | 2004-08-18 | Bi-recombinant active carrier vaccine of co-expression pig gyrate virus and foot and mouth disease virus |
Publications (2)
| Publication Number | Publication Date |
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| CN1749397A true CN1749397A (en) | 2006-03-22 |
| CN1749397B CN1749397B (en) | 2010-12-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200410011047 Expired - Fee Related CN1749397B (en) | 2004-08-18 | 2004-08-18 | Bi-recombinant active carrier vaccine of co-expression pig gyrate virus and foot and mouth disease virus |
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| CN (1) | CN1749397B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220375B (en) * | 2007-01-11 | 2011-07-27 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PT7.5N) |
| CN102861327A (en) * | 2012-09-27 | 2013-01-09 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yoke antibody and injection and freeze-dried powder containing same |
| CN103865955A (en) * | 2009-10-16 | 2014-06-18 | 佐蒂斯有限责任公司 | Infectious clones of Torque teno virus |
| CN110041409A (en) * | 2019-05-07 | 2019-07-23 | 山东省农业科学院畜牧兽医研究所 | A kind of saltant type porcine circovirus 2 type is viral and applies |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7276353B2 (en) * | 2001-12-12 | 2007-10-02 | Virginia Tech Intellectual Properties, Inc. | Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof |
-
2004
- 2004-08-18 CN CN 200410011047 patent/CN1749397B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220375B (en) * | 2007-01-11 | 2011-07-27 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PT7.5N) |
| CN103865955A (en) * | 2009-10-16 | 2014-06-18 | 佐蒂斯有限责任公司 | Infectious clones of Torque teno virus |
| CN102861327A (en) * | 2012-09-27 | 2013-01-09 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yoke antibody and injection and freeze-dried powder containing same |
| CN110041409A (en) * | 2019-05-07 | 2019-07-23 | 山东省农业科学院畜牧兽医研究所 | A kind of saltant type porcine circovirus 2 type is viral and applies |
| CN110041409B (en) * | 2019-05-07 | 2022-09-06 | 山东省农业科学院畜牧兽医研究所 | Mutant porcine circovirus type 2 virus and application thereof |
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| Publication number | Publication date |
|---|---|
| CN1749397B (en) | 2010-12-08 |
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