猪传染性胃肠炎病毒TH-98株HP基因的克隆与序列分析

Cloning and Sequence Analysis of Hp Gene from Transmissible Gastroenteritis Virus of Porcine Isolate TH-98

hp基因位于猪传染性胃肠炎病毒(TGEV)基因组的3′端,该基因在病毒复制时表达编码一个78AA、9100的疏水蛋白。该蛋白可以与猪超免抗TGEV血清发生免疫沉淀反应。目前,对其功能的研究还不是十分清楚。但就其在细胞内的位置而言,hp蛋白可能对复制复合体膜的缔合以及病毒粒子的装配起重要作用。根据Genebank已发表的TGEVhp基因cDNA序列,利用Oligo4.1软件设计一对引物,进行RT-PCR扩增hp基因。将PCR产物用KpnI和XbaI酶切后克隆到载体pPROEXHTc的KpnI和XbaI多克隆位点上。然后对重组质粒进行相应的酶切鉴定和PCR鉴定。将重组子测序并且与其国外分离株进行同源性比较。结果表明,hp基因克隆成功是可信的。本研究为进一步研究TGEV的基因组及其非结构蛋白奠定了重要基础。

:Hp gene encoding a 78 amino acid,9 100 hydrophobic protein (Hp) and, mapping at the 3′end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome was expressed during virus replication .the protein is a nonstructure protein .Hp was immunoprecipitable with porcine hyperimmune antiTGEV serum.The function of Hp is not clear now. The intracellular location of Hp suggests that it may play a role in the membrane association of replication complexes or in virion assembly.A pair of primers was designed to amplify hp gene by RTPCR according to the published sequence of TGEV 's hp gene cDNA with Oligo version 4.0software. The product of PCR was digested with KpnI and XbaI. The digested fragment was cloned in KpnI and XbaI multiple cloning sites of the pPROEX HTc vector .the recombinant pPROEX HTchp plasmid was identified and analyzed by corresponding restriction endonuclease and PCR. Recombinant pPROEX HTchp was sequenced and analyzed in comparison with the other homologous strains. the results affirmed that hp gene from TGEV of China isolate TH98 is extremely conservative.