抗人红细胞单链抗体与狂犬病毒G蛋白双功能融合蛋白的构建及生物学活性检测  

Construction and bioactivity detection on bifunctional fusion protein of anti-human red blood cell scFv and RV G

作　　者：周松峰[1] 廖文军 覃绍敏[1] 袁书智 白安斌[1] 吴健敏[1] 

机构地区：[1]广西兽医研究所广西畜禽疫苗重点实验室,南宁530001 [2]北京博莱得利生物技术有限责任公司,北京100850

出　　处：《中国人兽共患病学报》2012年第6期597-601,共5页Chinese Journal of Zoonoses

基　　金：广西科技厅基本科研业务费专项(桂科专项11-4)~~

摘　　要：目的为构建具有凝集性、免疫反应性的双功能融合蛋白。方法本研究利用特异性引物从重组质粒pMD-2E8scFv和pMD-G中以PCR方法扩增2E8基因(抗人红细胞H抗原单链抗体基因)和Kg基因(狂犬病毒G基因主要抗原表位区),采用重叠延伸(SOE)PCR法拼接成融合基因2E8Kg,构建原核表达质粒pET-Trx-2E8Kg并将其转化至BL21(DE3)pLysS中进行诱导表达并对诱导菌液进行SDS-PAGE分析。结果 2E8Kg融合基因获得了高效表达并主要以包涵体形式存在,表达量占菌体总蛋白的23.5%左右,分子量约为77.7kD,与预期的大小一致。将表达的蛋白以亲和层析法进行纯化并通过谷胱甘肽还原法进行复性,纯化后蛋白纯度达到98.9%。结论经Western-blot检测和红细胞凝集试验证明,2E8kg融合蛋白既能够与狂犬病毒(RV)高免血清发生特异性反应,又能够与人红细胞结合,具有双功能特性。The purpose of this study is to construct fusion proteins that can generate agglutination and immune reaction.Specific primers were used to amplify,from the recombinant plasmids of both pMD-2E8scFv pMD-G with PCR,2E8 gene（scFv gene against H antigen of human erythrocytes） and Kg gene（the major antigen epitope domain of G gene）.These two were thereupon spliced into a fusion gene by splicing overlap extension PCR to construct prokaryotic expression plasmid pET-Trx-2E8Kg,and was then transformed into competent cell BL21（DE3）pLysS.Finally,protein expression was induced with IPTG and analysed by SDS-PAGE.The results showed that the target fusion protein was successfully expressed and identified in inclusion bodies,with an expression accounting for 23.5% of the total bacterial protein.The molecular weight was 77.7 kD,consistent with the expected size.The purity of the expressed protein reached 98.9% after being purified by affinity chromatography and refolded by Glutathione reduction.The results of Western-blot detection and agglutination test on red blood cell indicated that the fusion protein 2E8kg had bifunctional characteristics,not only being able to have specific reaction with anti-rabies hyperimmune serum,but also could combine with human red blood cell.

关 键 词：狂犬病毒 G基因 单链抗体 双功能融合蛋白 原核表达 

分 类 号：R373[医药卫生—病原生物学]