柯萨奇病毒A6型VP1和VP2基因的原核表达及多克隆抗体制备  

Prokaryotic expression of coxsackie virus A6 genes and preparation of polyclonal antibody against the VP1 and VP2 protein

作　　者：陈静[1,2] 李鹏飞[2] 吴杰[2] 孟胜利[2] 申硕[2] 王泽鋆[2] Chen Jing;Li Pengfei;Wu Jie;Meng Shengli;Shen Shuo;Wang Zejun(China Three Gorges University,443002 Yichang,China;Viral Vaccine Research Laboratory, Wuhan Institute of Biological Products,430207 Wuhan,China)

机构地区：[1]三峡大学医学院,宜昌443002 [2]武汉生物制品研究所病毒性疫苗研究一室,430207

出　　处：《中华实验和临床病毒学杂志》2018年第6期640-645,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：三峡大学人才专项经费8000303(20171227);2017湖北省技术创新专项(重大项目);国家“重大新药创制”科技重大专项(2015ZX09102021).

摘　　要：目的构建柯萨奇病毒A6型(coxsackie virusA6,CVA6)VP1和VP2基因的原核表达载体,诱导表达重组蛋白,制备抗CVA6VP1和VP2兔抗血清。方法逆转录PCR扩增CVA6VP1和VP2基因片段,整合入表达载体pGEX-6p-1,转化至大肠杆菌BL21(DE3),异丙基硫代半乳糖苷(IPTG)诱导蛋白表达,经过大型SDS-PAGE制备电泳并进行切胶回收纯化,纯化后的融合蛋白GST-VP1和GST-VP2分别免疫家兔,获得兔抗血清。Westernblot(WB)和间接免疫荧光试验(indirectimmunofluorescence assay,IFA)鉴定抗体。结果CVA6VP1和VP2原核表达载体经PCR、双酶切和测序鉴定正确,获得了高纯度的CVA6VP1和VP2融合蛋白,WB和IFA鉴定多克隆抗体制备成功。结论成功构建了CVA6VP1和VP2原核表达载体,获得了重组的CVA6VP1和VP2蛋白及其多克隆抗体。Objective To construct the prokaryotic plasmid expressing the recombinant protein Coxsackie virus A6 VP1 or VP2 and prepare the antiserum of rabbit anti-CVA6 VP1 or anti-CVA6 VP2. Methods CVA6 VP1and VP2 gene fragments were amplified by reverse transcription PCR and inserted into the prokaryotic expression vectors. The recombinant plasmids were expressed in E. coli BL21 (DE3). After induction with Isopropyl β-D-Thiogalactoside (IPTG), the fusion proteins were obtained, and then purified by SDS-PAGE electrophoresis and gel extraction. The polyclonal antibodies were prepared by immunizing rabbits with the fusion proteins and analyzed with Western blot(WB) and indirect immunofluorescence assay(IFA). Results The prokaryotic expression vector of CVA6 VP1 or VP2 was confirmed by PCR, double enzyme digestion and sequencing. CVA6 VP1 and VP2 fusion proteins with high purity were obtained. WB and IFA were used to identify polyclonal antibodies. Conclusions The CVA6 VP1 and VP2 prokaryotic expression vectors were successfully constructed, and the recombinant CVA6 VP1 and VP2 proteins and their corresponding polyclonal antibodies were obtained.

关 键 词：柯萨奇病毒A6型 VP1 VP2 原核表达 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]