PRRSV Hn-1/06株GP5蛋白基因的克隆及表达载体的构建

目的]克隆PRRSVHn-1/06株GP5蛋白基因并构建其原核表达载体。方法]根据PRRSV美洲株ATCC-VR2332的ORF5基因序列设计特异性引物,用RT-PCR方法从Hn-1/06株中扩增得到GP5蛋白基因的片段,与PTG19-T载体连接获得其阳性克隆PTG19-T-ORF5。以该基因片段为模板分别设计ORF5完整序列和删除信号肽的ORF5序列两条引物,进行PCR扩增,将目的片段与原核表达载体pET32a连接,并对其进行酶切鉴定。结果]扩增得到GP5蛋白基因全长序列603 bp,编码200个氨基酸残基;具有3个潜在的N-糖基化位点和9个半胱氨酸;分子量为22.4 kD,等电点为8.550;双酶切pET32a-ORF5鉴定,结果与预期一致,证实重组质粒构建成功。结论]成功构建了PRRSVHn-1/06株GP5蛋白基因的表达载体,为深入研究GP5蛋白的本质与功能及其致病性奠定基础。

Cloning and Construction of Expression Vector of GP5 Gene of PRRSV Hn-1/06 Strain

Objective] The aim was to clone the GP5 gene of PRRSV Hn-1/06 strain and construct its prokaryotic expression vector.Method] The fragment of GP5 gene was amplified from the Hn-1/06 strain by RT-PCR using the specific primers designed from the sequence of the ORF5 gene from PRRSV American strain ATCC-VR2332.The positive clone of PTG19-T-ORF5 was obtained after connected with PTG19-T vector.The obtained fragment of the gene was used as template to design 2 primers with the complete sequence of ORF5 and the sequence of ORF5 deleting the signal peptide resp.The target fragment was connected with the pET32a prokaryotic expression vector after PCR amplification,then identified with enzyme restriction.Result] The GP5 gene with full length sequence of 603 bp was obtained by amplification and it encoded 200 amino acid residues and had 3 potential N linked glycosylation sites and 9 cysteines.Its molecular weight was 22.4 kD and isoelectric point was 8.550.The result of two enzyme restriction identified pET32a-ORF5 was consistent with the anticipating result,which confirmed the recombinant plasmid was constructed successfully.Conclusion] The expression vector of GP5 gene of PRRSV Hn-1/06 strain was constructed successfully,which laid the foundation for the further study of the essence,function and the pathogenicity of GP5 protein.