HV单链抗体/鱼精蛋白片段融合蛋白1A8 ScFv/Cκ/P的构建和表达  

Construction and expression of a single-chain HV antibody-protamine fragment fusion protein 1A8 ScFv/Cκ/P

作　　者：陈锐[1] 杨洁[1] 张芳琳[2] 秦炜炜[1] 王涛[1] 张瑞[1] 孟艳玲[3] 胡刚[2] 徐志凯[2] 杨安钢[3] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033 [2]第四军医大学基础部微生物学教研室,陕西西安710033 [3]第四军医大学基础部免疫学教研室,陕西西安710033

出　　处：《第四军医大学学报》2008年第11期967-970,共4页Journal of the Fourth Military Medical University

基　　金：国家973计划项目(2004CB518805);军队"十一五"科技攻关项目(06G084)

摘　　要：目的:构建汉坦病毒(Hantavirus,HV)单链抗体/鱼精蛋白片段融合蛋白1A8 ScFv/Cκ/P基因的原核表达载体,蛋白经表达后纯化并进行1A8 ScFv/Cκ/P融合蛋白的功能活性鉴定.方法:采用PCR的方法,扩增单链抗体基因1A8 ScFv和1A8 ScFv/Cκ,将鱼精蛋白片段的编码序列设计入扩增引物,获得HV单链抗体/鱼精蛋白片段的融合蛋白基因1A8 ScFv/Cκ/P.将获得的重组基因克隆入原核表达载体pET32a,并在大肠杆菌BL21中表达.表达产物经SDS-PAGE和Western Blot鉴定,用Ni-NTA螯合层析介质进行纯化.采用ELISA方法分析1A8 ScFv/Cκ/P融合蛋白与HV抗原的结合活性,凝胶迁移阻滞实验检测1A8 ScFv/Cκ/P融合蛋白与质粒DNA的结合活性.结果:成功构建了HV 1A8 scFv/Cκ/P融合蛋白基因的表达载体,在大肠杆菌中实现了可溶性表达.通过功能活性测定,纯化的1A8 scFv/Cκ/P融合蛋白既保持了与HV抗原的结合能力,同时又具有与核酸的结合活性.结论:构建和表达的HV 1A8 scFv/Cκ/P融合蛋白具有HV抗原结合活性和DNA结合的活性.AIM ： To construct, express and purify a fusion protein, which is composed of a single-chain antibody fragment （ 1 A8 ScFv） that is recognized as Hantavirus （HV） nucleocapsid protein, a constant region of light chain （Cκ） and a truncated protamine, and to analyze the binding activity of the fusion protein. METHODS： Pairs of oligonucleotide primers were designed and used to amplify the 1A8 ScFv and the 1A8 ScFv/Cκ. The truncated protamine fragment coding sequence was added in the 3＇ terminus primer. The fusion protein gene 1A8 ScFv/Cκ/P was then cloned into expression vector pET32a and expressed in E. coli BL21 （DE3）. Expressed protein was detected by SDS-PAGE and Western Blot and purified by Ni-NTA chelating agarose. The anti- gen-binding activity of the 1AS ScFv/Cκ/P fusion protein was confirmed by ELISA, and the DNA binding ability was confirmed by electrophoretic mobility shift assay （EMSA）. RESULTS： HV 1A8 ScFv/Cκ/P encoding sequence was correctly cloned into the expression vector. The recombinant protein was solubly expressed in E. coli BL21 induced by IPTG. ELISA confirmed that it had specific antigen binding activity ; EMSA assured that it had DNA binding activity. CONCLUSION： The 1A8 ScFv/Cκ/P fusion protein can specially bind with both HV antigen and DNA.

关 键 词：汉坦病毒 单链抗体 鱼精蛋白 融合蛋白 靶向输送 

分 类 号：R392.116[医药卫生—免疫学]