猪繁殖与呼吸综合征病毒BJ-4株GP4蛋白的原核表达与纯化  

Prokaryotic expression and purification of GP4 protein of porcine reproductive and respiratory syndrome virus BJ-4 strain

作　　者：车志芬 史西保[2] 张改平[1,3] 扣莉云 周景明[1] 祁艳华[1] 王爱萍[1] 

机构地区：[1]郑州大学生命科学学院,河南郑州450001 [2]河南师范大学生命科学学院,河南新乡453007 [3]河南农业大学牧医工程学院,河南郑州450002

出　　处：《西北农林科技大学学报（自然科学版）》2017年第5期7-14,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金面上项目(31472177)

摘　　要：【目的】原核表达和纯化猪繁殖与呼吸综合征病毒(PRRSV)的结构蛋白GP4,为深入了解其结构和功能奠定基础。【方法】采用RT-PCR方法扩增PRRSV BJ-4株ORF4基因片段,将其克隆到pMD19-T载体中并进行测序。从测序正确的重组质粒pMD19-T-ORF4中扩增缺失N端及C端疏水序列的基因片段tGP4(truncated GP4),再将其亚克隆到pET-32a载体并转化到大肠杆菌BL21,IPTG进行诱导表达。对表达的重组蛋白进行可溶性分析,使用尿素梯度法进行蛋白纯化,并通过ELISA法检测重组蛋白的免疫活性。【结果】RT-PCR获得了537bp的ORF4。成功构建了原核表达载体pET-32a-tGP4,在IPTG诱导后重组蛋白以包涵体的形式大量表达。使用尿素梯度法获得了纯度较高的目的蛋白,纯化的目的蛋白能与PRRSV阳性血清特异性结合。【结论】使用原核细胞成功表达了PRRSV的结构蛋白GP4,纯化后的重组蛋白具有很好的免疫原性。[Objective] Prokaryotic expression and purification of GP4 protein of porcine reproductive and respiratory syndrome virus （PRRSV） were conducted to provide basis for understanding the structure and function of PRRSV GP4. [Method] In this study,the ORF4 gene of PRRSV BJ-4 strain was amplified by RT-PCR and was cloned into the vector pMD19-T. Then, the truncated ORF4 without N and C termi- nals was amplified by PCR and sub-cloned to pET-32a. The pET-32a-tGP4 was transformed into E. coli BL21（DE3） and induced by IPTG. The solubility of recombinant protein was analyzed and it was purified using urea gradient method. Finally,the recombinant protein immune activity was detected by ELISA. [Re-sultl The ORF4 was 537 bp by RT-PCR. The recombinant plasmid pET-32a-tGP4 was constructed suc- cessfully. The induction experiment showed that E. coli could express the recombinant protein GP4 in in- clusion body form. Highly pure inclusion body was obtained using urea gradient method and the purified re- combinant protein could react well with PRRSV polyclonal anti-serum. [Conclusion] The GP4 protein of PRRSV was successfully expressed using prokaryotic cells and the purified recombinant protein had good immunogenicity.

关 键 词：猪繁殖与呼吸综合征病毒 GP4 原核表达 蛋白纯化 

分 类 号：S858.28[农业科学—临床兽医学]