A群猪轮状病毒OSU株VP7基因的克隆及其在杆状病毒中的表达
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摘要
猪轮状病毒(Porcine Rotavims，PRV)属弧肠孤病毒科轮状病毒属成员，是引起仔猪病毒性腹泻的主要病原之一。该病幼畜最为易感，1-4周龄仔猪发病率超过80％，死亡率7-20％，且症状严重，主要表现为严重腹泻，部分病例因严重脱水、酸碱平衡失调、继发感染而死亡。快速准确的诊断和有效免疫预防是本病防制的主要内容。病毒外膜的VP7糖蛋白是RV的主要免疫保护性抗原，由其刺激机体产生的抗体对中和病毒、消除感染起主要作用。因此，该基因的克隆及表达，对于制备新型疫苗和特异性诊断抗原具有重要意义。
本研究用恒河猴胚肾细胞(MA-104细胞系)繁殖并扩增了A群PRV G5型标准毒株OSU。根据Genebank已发表的PRV VP7基因cDNA序列，利用Oligo4.1软件设计并合成一对引物，通过反转录—聚合酶链反应(RT—PCR)扩增出长约1.0kb的基因片段，命名为VP7。将其克隆至PMD-18T载体，构建了重组质粒PMD-18T-VP7。对PMD-18T-VP7中的VP7进行序列测定，并将测序结果同仔猪多发的其它四个G血清型代表毒株(G9型ICB2185株、G10型P343株、G4型Gottfried株、G2型—C134株)及OSU-1株(参考序列)的VP7基因进行了序列比较。与OSU-1株的核苷酸和氨基酸同源性分别为99.4％和99.7％。与其它四个毒株的核苷酸同源性分别为77.9％、71.7％、75.3％、87.7％；推导的编码氨基酸同源性分别为83.1％、87.1％、84.7％、96.3％。
根据测序结果设计并合成另一对引物，将该基因克隆至真核表达载体PblueBAChisC的BglⅡ和HindⅢ之间的多克隆位点上，命名为重组表达载体PBC-VP7。酶切、PCR及套式PCR鉴定结果表明获得了PRV-VP7基因与PblueBAChisC质粒的阳性重组子。纯化该重组质粒并与线性杆状病毒DNA Bac-N-Blue共转染昆虫细胞sr9，5d后收获重组病毒。重组杆状病毒DNA分子的PCR及酶切鉴定表明，获得了PRV-VP7基因与杆状病毒DNA的重组子，命名为A-1代病毒。将经3次蚀斑筛选纯化后的A-1代病毒接种sf9细胞大量繁殖后收获，PCR鉴定为完全纯化的病毒命名为A-2代病毒。接种A-2代病毒于sf9单层细胞上，4d后收获病变细胞，通过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫酶斑点技术(Dot-ELISA)分析，表明PRV-VP7基因在杆状病毒表达系统中得到了表达。VP7基因的真核表达，为进一步研究PRV VP7蛋白的结构和功能，研制PRV基因工程疫苗以及制备特异性诊断抗原奠定了基础。
Porcine Rotavirus (PRV) belongs to the family reovindae. It is one of the major pathogens
that cause life threatening diarrhea in piglets. This kind of diadrrhea was the easiest infected by
young animal. Its infectious ratio is over 80%, and its mortality in pigS 1 to 4 weeks is 20%.
Rotavirus diarrhea's typically clinical manifestation is seriously diarrhea. Partial infected pigletS
were deaded due to severe Ioss of water, imbalance of acid and alkaIi and secondary infection.
Accurate diagnosis and effectiv Veccination are main means for the prevention of Porcine
diarrha ReSearh showed tha the outer caPsid glycoprotein VP7 of PRV particle is major
protective anigen. It can make body generating the antibody to neutralize rotavirus and
eldriinate infechon. So gaining VP7 gene and itS exPressing product is key to the preparaion of
the new tyPe vaccine and sPeCific diagnOsis anigen.
In this StLIdy, G5 serotyPe A gIDuP POreine rotavha stanha strain OSU was propagated
on MA-l04 monolayer cell. A Pair of prirners was designed to amplify VP7 gene by RT-PCR
according to the published Sequence of PRVs VP7 gene with Oligo4. l software. The product of
PCR named VP7 is aPprotriate l.0kb in length. The VP7 gene was cloned into the vector
PMD-l8T and the recombinan was named PMD-l8T-VP7. Identifications of restriction
empme, PCR and sequencing inditaled tha the VP7 gene has been cloned successfully
PMD-18T-VP7 was analpe in comParison with the other fOur G serotyPe PRV strain wn
genes, Gene sequence comparison indibed that OSU shad 99.4%, 77.9%, 71 .7%, 75.3% and
87.7% identities with OSU-1 (G5), ICB2l85 (G9), P343 (G10), Gottiffied (G4) and CI34 (G2)
resPectively The deduced arnino acid Sequences were 99.4%, 83. l %, 87. l%, 84.7% and 96.3%
cormsPOndingly The results affinned that VP7 gene of PRV strain OSU was not conservative.
mp was insertd in Bgl IIand HindIII multiple cloning sites of the vector PblueBAChisC
using Another pair ofpriIners designed accoIding to the sequence of VP7. PblueBAChisC-VP7
was identified and analped by compnding restriction endonuclease, PCR and nested PCR
on the basis of the genetic siteS of PblueBAChisC, which was idenhfied as VP7 gene of PRV
hafied PblueBAChisC-VP7 and BarmidN-blue DNA were cbeinfected insect cell sro
and harvested Mmbinan beculovirus 5d later Identification with restriction empme and
PCR showed VP7 gene has been arembwt comehy with baculovirus DNA and namd
A-l. A-l was infected on monolayer sffi ther the third plaque purification to reproduce in
quantity and named it A-2. After A-2 was infeCtal on sffi monolayer cell 4d harvested the CPE
cell. The analysis resultS with SDS-PAGE and Dot-ELISA showed that PRV VP7 gene was
-- o --
Abstract
expressed in baculovirus.
the eukaryotic expression of PRV VP7 gene provided not only the important basis for further research of the structure and function of the PRV VP7 protein and the preparation of vaccine, but an new viral diagnosis method.
Postgraduate: XU Hongjun Major: Preventive Veterinary Science Supervisor: Prof. WANG Junwei Prof. LI Yijing
本研究用恒河猴胚肾细胞(MA-104细胞系)繁殖并扩增了A群PRV G5型标准毒株OSU。根据Genebank已发表的PRV VP7基因cDNA序列，利用Oligo4.1软件设计并合成一对引物，通过反转录—聚合酶链反应(RT—PCR)扩增出长约1.0kb的基因片段，命名为VP7。将其克隆至PMD-18T载体，构建了重组质粒PMD-18T-VP7。对PMD-18T-VP7中的VP7进行序列测定，并将测序结果同仔猪多发的其它四个G血清型代表毒株(G9型ICB2185株、G10型P343株、G4型Gottfried株、G2型—C134株)及OSU-1株(参考序列)的VP7基因进行了序列比较。与OSU-1株的核苷酸和氨基酸同源性分别为99.4％和99.7％。与其它四个毒株的核苷酸同源性分别为77.9％、71.7％、75.3％、87.7％；推导的编码氨基酸同源性分别为83.1％、87.1％、84.7％、96.3％。
根据测序结果设计并合成另一对引物，将该基因克隆至真核表达载体PblueBAChisC的BglⅡ和HindⅢ之间的多克隆位点上，命名为重组表达载体PBC-VP7。酶切、PCR及套式PCR鉴定结果表明获得了PRV-VP7基因与PblueBAChisC质粒的阳性重组子。纯化该重组质粒并与线性杆状病毒DNA Bac-N-Blue共转染昆虫细胞sr9，5d后收获重组病毒。重组杆状病毒DNA分子的PCR及酶切鉴定表明，获得了PRV-VP7基因与杆状病毒DNA的重组子，命名为A-1代病毒。将经3次蚀斑筛选纯化后的A-1代病毒接种sf9细胞大量繁殖后收获，PCR鉴定为完全纯化的病毒命名为A-2代病毒。接种A-2代病毒于sf9单层细胞上，4d后收获病变细胞，通过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫酶斑点技术(Dot-ELISA)分析，表明PRV-VP7基因在杆状病毒表达系统中得到了表达。VP7基因的真核表达，为进一步研究PRV VP7蛋白的结构和功能，研制PRV基因工程疫苗以及制备特异性诊断抗原奠定了基础。
Porcine Rotavirus (PRV) belongs to the family reovindae. It is one of the major pathogens
that cause life threatening diarrhea in piglets. This kind of diadrrhea was the easiest infected by
young animal. Its infectious ratio is over 80%, and its mortality in pigS 1 to 4 weeks is 20%.
Rotavirus diarrhea's typically clinical manifestation is seriously diarrhea. Partial infected pigletS
were deaded due to severe Ioss of water, imbalance of acid and alkaIi and secondary infection.
Accurate diagnosis and effectiv Veccination are main means for the prevention of Porcine
diarrha ReSearh showed tha the outer caPsid glycoprotein VP7 of PRV particle is major
protective anigen. It can make body generating the antibody to neutralize rotavirus and
eldriinate infechon. So gaining VP7 gene and itS exPressing product is key to the preparaion of
the new tyPe vaccine and sPeCific diagnOsis anigen.
In this StLIdy, G5 serotyPe A gIDuP POreine rotavha stanha strain OSU was propagated
on MA-l04 monolayer cell. A Pair of prirners was designed to amplify VP7 gene by RT-PCR
according to the published Sequence of PRVs VP7 gene with Oligo4. l software. The product of
PCR named VP7 is aPprotriate l.0kb in length. The VP7 gene was cloned into the vector
PMD-l8T and the recombinan was named PMD-l8T-VP7. Identifications of restriction
empme, PCR and sequencing inditaled tha the VP7 gene has been cloned successfully
PMD-18T-VP7 was analpe in comParison with the other fOur G serotyPe PRV strain wn
genes, Gene sequence comparison indibed that OSU shad 99.4%, 77.9%, 71 .7%, 75.3% and
87.7% identities with OSU-1 (G5), ICB2l85 (G9), P343 (G10), Gottiffied (G4) and CI34 (G2)
resPectively The deduced arnino acid Sequences were 99.4%, 83. l %, 87. l%, 84.7% and 96.3%
cormsPOndingly The results affinned that VP7 gene of PRV strain OSU was not conservative.
mp was insertd in Bgl IIand HindIII multiple cloning sites of the vector PblueBAChisC
using Another pair ofpriIners designed accoIding to the sequence of VP7. PblueBAChisC-VP7
was identified and analped by compnding restriction endonuclease, PCR and nested PCR
on the basis of the genetic siteS of PblueBAChisC, which was idenhfied as VP7 gene of PRV
hafied PblueBAChisC-VP7 and BarmidN-blue DNA were cbeinfected insect cell sro
and harvested Mmbinan beculovirus 5d later Identification with restriction empme and
PCR showed VP7 gene has been arembwt comehy with baculovirus DNA and namd
A-l. A-l was infected on monolayer sffi ther the third plaque purification to reproduce in
quantity and named it A-2. After A-2 was infeCtal on sffi monolayer cell 4d harvested the CPE
cell. The analysis resultS with SDS-PAGE and Dot-ELISA showed that PRV VP7 gene was
-- o --
Abstract
expressed in baculovirus.
the eukaryotic expression of PRV VP7 gene provided not only the important basis for further research of the structure and function of the PRV VP7 protein and the preparation of vaccine, but an new viral diagnosis method.
Postgraduate: XU Hongjun Major: Preventive Veterinary Science Supervisor: Prof. WANG Junwei Prof. LI Yijing
引文
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