猪繁殖和呼吸综合征病毒福建毒株ORF5的序列分析及原核表达

根据猪繁殖和呼吸综合征病毒(PRRSV)已发表的核苷酸序列,设计了一对特异性引物(p1/p2),用RT-PCR扩增PRRSV福建毒株FJ-1的全长糖基化囊膜蛋白基因(ORF5),将扩增产物连接到pUCm-T载体并转化大肠杆菌DH10B,筛选阳性重组质粒进行序列测定与分析.再设计另一条引物(p3)与p2从重组载体pUCm-T-ORF5中扩增出缺失了N端30个氨基酸残基的tGP5的编码序列tORF5,克隆入表达载体pGEX-4T-3后在大肠杆菌中表达.结果表明,FJ-1毒株ORF5基因为603 bp,编码200个氨基酸,与北美型参考毒株VR-2332、CH-1a及欧洲型参考毒株LV的氨基酸同源性分别为87%、89%和53%,推定福建毒株FJ-1属于北美型毒株.原核表达产物经过SDS-PAGE及Western Blot分析,证实为GP5与谷胱甘肽转移酶(GST)的融合蛋白.分子量为41 kDa.表达量约占菌体蛋白的15%,主要以包涵体形式存在.

Cloning, Sequence Analysis and Prokaryotic Expression of ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Strain FJ-1

The full-length of ORF5 gene encoding the envelope glycoprotein(GP5) of porcine reproductive and respiratory syndrome virus strain FJ-1 was amplified by RT-PCR with a pair of primers(p1/p2) and cloned into plasmid pUCm-T.The recombinant plasmid designated as pUCm-T-ORF5 was subjected to sequencing and analysis and used as the template to amplify the truncated ORF5(tORF5),which is coding the GP5 lacking the first 30 residues at the amino terminal region,by the use of primers(p2/p3).The tORF5 gene was consequently cloned into the vector of pGEX-4T-3 for prokaryotic expression in E.coli.The result showed that the ORF5 gene of FJ-1 was 603 bp in full-length and encodes 200 amino acids,it shared 87%,89% and 53% amino acid homology with strains of VR-2332,CH-1a(North American genotype) and LV(European genotype),respectively.Based on these results,it was supposed that the FJ-1 isolate belongs to the North American genotype.The expression of tGP5 in the E.coli BL21(ED3) was confirmed by SDS-PAGE and western-blot,and the molecular weight of the expressed protein was approximately 41 kDa and mainly in the form of inclusion body.