人抗HBsAg单链抗体/鱼精蛋白截短体融合蛋白基因的构建、表达及活性鉴定  

Construction and expression of human anti-HBsAg ScFv/tP fusion protein in E.coli and identification of its binding activity

作　　者：孟艳玲[1] 温伟红[1] 薛茜[1] 张勇[1] 张巍[1] 鲍炜[1] 任君琳[1] 贾林涛[2] 王成济[2] 杨安钢[1] 

机构地区：[1]第四军医大学基础部免疫学教研室,陕西西安710033 [2]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

出　　处：《第四军医大学学报》2006年第15期1349-1352,共4页Journal of the Fourth Military Medical University

基　　金：国家重点基础研究发展(973)计划(2004CB518805);国家自然科学基金(30400379)

摘　　要：目的:构建人抗HBsAg单链抗体(ScFv)/鱼精蛋白截短体(truncated protam ine,tP)融合基因,在大肠杆菌中进行表达纯化并分析ScFv/tP融合蛋白的活性.方法:设计引物扩增人抗HBsAg ScFvHBs15基因,在其3′端引物引入tP的编码基因,PCR扩增获得ScFv/tP融合基因,将其克隆入原核表达载体pET-32 a后,在大肠杆菌BL2 l(DE3)LysS内诱导表达.表达产物经SDS-PAGE及W estern b lot鉴定后,用N i-NTA螯合层析介质纯化.间接ELISA分析ScFv/tP融合蛋白的亲和活性,凝胶迁移阻滞实验检测ScFv/tP融合蛋白与DNA结合的情况.结果:成功获得人抗HBsAg ScFv/tP融合基因,经IPTG诱导后在大肠杆菌中表达,表达的ScFv/tP融合蛋白不仅保持了与HBsAg结合的能力,同时具有DNA结合活性.结论:ScFv与tP融合后,同时具有与抗原和DNA结合的活性,为该ScFv的进一步应用奠定了基础.AIM： To construct a fusion gene of human anti-HBsAg ScFv/tP and analyze the binding activity of ScFv/tP fusion protein with the antigen and DNA ＂after the protein was expressed and purified in E. coli. METHODS： Three oligonucleotide primers were designed and used to amplify the ScFv HBs15 gene. Coding sequence of tP was added in the 3＇ primer terminus. The ScFv/tP gene was amplified by PCR and cloned into expression vector pET-32a 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 antigen-binding activity of the ScFv/tP fusion protein was confirmed by indirect ELISA, and the DNA binding ability was confirmed by EMSA. RESULTS： Restriction endonuclease digestion and DNA sequencing proved that ScFv/tP gene was correctly cloned into expression vector, SDS-PAGE and Western blot analysis showed that ScFv/tP fusion protein was successfully expressed in E. coli BL21. Indirect ELISA assured that the fusion protein had antigen-binding activity, and EMSA confirmed that it had specific DNA binding activity. CONCLUSION： The anti-HBsAg ScFv/tP fusion protein expressed in E.coli could specially bind with both HBsAg and DNA fragment.

关 键 词：单链抗体 肝炎表面抗原 乙型 鱼精蛋白类 

分 类 号：R392.11[医药卫生—免疫学]