畜牧与兽医2025，Vol.57Issue(8)：108-114,7.
猪繁殖与呼吸综合征病毒非结构蛋白NSP9的表达纯化及多克隆抗体的制备
Expression and purification of PRRSV non-structural protein NSP9 and preparation of its polyclonal antibody
摘要
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 kDa in size,and its prepared polyclonal antibody against PRRSV possessed good biological activity.The results of this experiment laid an important foun-dation for the development of PRRSV detection and immunological serodiagnostic reagents and for further research on the biological function of PRRSV NSP9.关键词
猪繁殖与呼吸综合征病毒/NSP9/原核表达/蛋白纯化/多克隆抗体Key words
PRRSV/NSP9/prokaryotic expression/protein purification/polyclonal antibodies分类
农业科技引用本文复制引用
刘凯,冯康,熊江广,王文清,张志榜,李鹏成,张锦华,张晓燕..猪繁殖与呼吸综合征病毒非结构蛋白NSP9的表达纯化及多克隆抗体的制备[J].畜牧与兽医,2025,57(8):108-114,7.基金项目
国家自然科学基金项目(31960697,32260880) （31960697,32260880）
江西省"双千计划"项目(jxsq2019101056) （jxsq2019101056）
