【摘要】 旨在获得猪繁殖与呼吸综合征病毒(PRRSV)的非结构蛋白NSP9及制备PRRSV NSP9的多克隆抗体。本研究应用PCR技术扩增出全长NSP9目的基因，将其克隆至原核表达载体pET-28a中，同时插入His和Flag融合标签，获得重组质粒pET-28a-NSP9;然后将重组质粒转化至BL21感受态细胞，经IPTG诱导并优化诱导温度和时间，SDS-PAGE和Western blot鉴定，观察NSP9蛋白的表达情况，确定NSP9蛋白的最佳表达条件，应用镍柱亲和层析纯化NSP9蛋白；最后将纯化后的NSP9蛋白免疫BALB/c小鼠，获得多克隆抗体，用ELISA测定抗体效价，Western blot鉴定抗体反应原性。结果：含有pET-28a-NSP9重组质粒的大肠杆菌，在IPTG终浓度为0.2 mmol/L诱导，16℃培养32 h的条件下，NSP9蛋白主要以可溶性形式表达，经镍柱亲和层析纯化成功获得高纯度的NSP9蛋白，经间接ELISA和Western blot测定成功获得特异性的NSP9多克隆抗体。综上，本研究以全长NSP9基因为模板，克隆转化后表达出72 kDa左右的可溶性重组蛋白，利用其作为抗原免疫制备的抗PRRSV多克隆抗体具备良好的生物活性，为后续PRRSV检测、免疫血清诊断试剂的研发及研究PRRSV NSP9的生物学功能奠定了重要基础。更多还原

【Abstract】 The aim of this study was to obtain a nonstructural protein NSP9 of porcine reproductive and respiratory syndrome virus(PRRSV) and to prepare polyclonal antibodies against PRRSV NSP9. Firstly, the full-length NSP9 target gene was amplified by PCR and was cloned into the prokaryotic expression vector pET-28a. Then, the His and Flag fusion tags were inserted at the same time to obtain the recombinant plasmid pET-28a-NSP9. Next, the recombinant plasmid was transformed into BL21 receptor cells, and was identified by IPTG induction, optimization of induction temperature and time, and by SDS-PAGE and Western blot. Thirdly, the expression of NSP9 protein was observed to determine the optimal expression conditions of NSP9 protein. Nickel column affinity chromatography was employed to purify NSP9 protein. Finally, the purified NSP9 protein was immunized into BALB/c mice to obtain polyclonal antibodies, and the antibody potency was determined by ELISA and antibody reactivity was identified by Western blot. The results showed that E.coli containing pET-28a-NSP9 recombinant plasmids was induced by final concentration of 0. 2 mmol/L IPTG and cultured at 16 ℃ for 32 h. The NSP9 protein was mainly expressed in a soluble form. High purity NSP9 protein was successfully obtained by nickel column affinity chromatography purification. The specific NSP9 polyclonal antibody was also successfully obtained by indirect ELISA and Western blot. In conclusion,in the present study,the full-length NSP9 gene was sued as a template,cloned and transformed to express a soluble recombinant protein of about 72 kD a in size,and its prepared polyclonal antibody against PRRSV possessed good biological activity. The results of this experiment laid an important foundation for the development of PRRSV detection and immunological serodiagnostic reagents and for further research on the biological function of PRRSV NSP9.更多还原