PRRSV BJ-4株ORF5基因的原核表达与重组蛋白的纯化  

Prokaryotic Expression of ORF5 Gene of PRRS Virus BJ-4 and Purification of Recombinant Protein

作　　者：谷红[1] 杨汉春[1] 郭鑫[1] 陈艳红[1] 

机构地区：[1]中国农业大学农业部预防兽医学重点开放实验室,北京100094

出　　处：《畜牧兽医学报》2004年第1期64-69,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：霍英东青年教师基金(71032)

摘　　要：为成功表达猪繁殖与综合征病毒(PRRSV)结构蛋白GP5,通过PCR方法从重组质粒pGEM ORF5扩增得到缺失N端疏水序列的基因片段dORF5(deletingORF5)。将dORF5克隆至原核高效表达载体pGEX 4T 2,在E.coliBL21细胞中成功表达了重组蛋白GST dORF5,表达产物以包涵体的形式存在,表达量为20 8%。Western Blot结果表明重组蛋白可被PRRSV阳性血清所识别。利用融合肽进行亲和层析得到高纯度的重组蛋白,为进一步研究PRRS病毒结构蛋白的结构和功能奠定了基础。In order to successfully express the structural protein GP5 of porcine reproductive and respiratory syndrome virus(PRRSV),the gene segment dORF5 deleting N-terminal very hydrophobic sequence were successfully amplified from recombinant plasmid pGEM-ORF5 by PCR and were cloned into prokaryotic expression vector pGEX-4T-2.The recombinant fusion proteins GST-dORF5 were highly expressed in E coli.cell BL21 in the forms of inclusion bodies and could amount to 20.8% of the total mass of bacterial proteins.Western-Blot showed the recombinant protein could react with the porcine polyclonal antibodies against PRRSV.Recombinant proteins were purified and renatured.The purity of recombinant proteins affinity-purified using Glutathione Sepharose 4B were above 90%.The studies provided fundamental data and materials for the further study on the structure and function of structural proteins of PRRSV.

关 键 词：PRRSV BJ-4株 ORF5基因 原核表达 重组蛋白 分离纯化 猪 繁殖与呼吸综合症 

分 类 号：S858.28[农业科学—临床兽医学]