PRRSV陕西分离株ORF5基因克隆、序列分析及原核表达  

Cloning,Sequence Analysis and Prokaryotic Expression of ORF5 Gene of PRRSV Shaanxi Strain

作　　者：王志昇[1] 许信刚[1] 童德文[1] 邢福珊[1] 陈曦[1] 唐麒麟[1] 宁蓬勃[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农业学报》2010年第8期1-6,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：陕西省科技攻关项目(2009K02-01);国家大学生创新性实验计划项目;教育部新世纪优秀人才支持计划项目(NCET-07-0701)

摘　　要：克隆猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因,并进行序列分析及原核表达。根据GenBank公布的PRRSV ORF5基因的核苷酸序列,设计并合成一对特异性引物,用RT-PCR方法扩增PRRSV陕西分离株糖基化囊膜蛋白基因ORF5,将其克隆入pGEM-T载体中,进行测序及序列分析。再设计另外1对引物扩增ORF5缺失编码N端31个氨基酸残基的基因片段,将截短的ORF5基因亚克隆入原核表达载体pET-32a中,在大肠杆菌BL21中进行原核表达。结果扩增到603 bp的PRRSV全长ORF5基因,序列分析表明,分离株与北美型代表株VR-2332和欧洲型代表株LV氨基酸同源性分别为87.8%和54.5%。因此推测陕西分离株属于北美型。SDS-PAGE可检测到大小约为38 ku的融合蛋白,主要以可溶性蛋白形式存在。West-ern-blot分析表明,重组蛋白可被PRRSV阳性血清所识别。According to the published complete nucleotide sequence of PRRSV（porcine reproductive and respiratory syndrome virus）in GenBank,a pair of primers specific to the full-length of ORF5 gene encoding the envelope glycoprotein GP5 was designed.The ORF5 gene fragment was amplified by RT-PCR and cloned into pGEM-T vector.The recombinant plasmid was sequenced and ORF5 gene was compared with GeneBank＇s other PRRSV strains.The ORF5 gene segment lacking the first 31 residues at the amino terminal region was amplified from recombinant plasmid by PCR,then the gene was cloned into vector pET-32a for prokaryotic expression in E.coli.The ORF5 gene of Shaanxi strain was 603 bp in full-length and encoded 200 amino acids,it shared 87.8 % and 54.5 % amino acid homology with strains of VR-2332（North American genotype） and LV（European genotype） respectively.The recombinant fusion proteins were highly expressed in E.coli BL21 in the forms of soluble protein.The molecular weight of the expressed fusion protein was approximately 40 ku.Western-blot showed that the recombinant protein can be recognized by PRRSV-positive serum.These results would lay a foundation for developing PRRS diagnostic antigen.

关 键 词：猪繁殖与呼吸综合征病毒 陕西分离株 ORF5 序列分析 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]