人源抗HER2单链抗体/轻链恒定区/鱼精蛋白截短体融合蛋白的基因构建、表达及活性鉴定  

Construction and expression of human anti-HER2 ScFv/Ck/tP fusion protein in E.coli and identification of its acti-vity

作　　者：王凯[1] 温伟红[2] 王涛[1] 张瑞[1] 秦炜炜[1] 雷小英[1] 杨安钢[2] 

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免疫学教研室,陕西西安710033

出　　处：《第四军医大学学报》2007年第7期581-584,共4页Journal of the Fourth Military Medical University

基　　金：国家重点基础研究发展(973)计划项目(2004CB518805);国家自然科学基金(30370314;30400379)

摘　　要：目的:构建人抗HER2单链抗体(ScFv)/人轻链恒定区(Ck)/鱼精蛋白截短体(tP)融合蛋白基因,在大肠杆菌中表达、纯化并分析该融合蛋白的活性.方法:设计引物扩增人抗HER2单链抗体e23sFv基因和轻链恒定区编码序列(Ck),连接成ScFv/Ck片段,人工合成tP序列,连接于ScFv/Ck末端,构建成ScFv/Ck/tP融合基因,将其克隆入原核表达载体pET32a后,在大肠杆菌BL21(DE3)LysS中诱导表达,表达产物经SDS-PAGE和Western Blot鉴定后,用Ni-NTA螯合层析介质纯化.细胞ELISA分析ScFv/Ck/tP融合蛋白抗原亲合活性,凝胶迁移实验检测ScFv/Ck/tP融合蛋白与DNA的结合活性.结果:成功构建了人抗HER2ScFv/Ck/tP融合基因,经IPTG诱导后在大肠杆菌中实现了可溶性表达.表达的ScFv/Ck/tP融合蛋白保持了与抗原的结合活性,同时具有结合DNA的能力.结论:ScFv与Ck,tP融合后,同时具有抗原和DNA结合活性,为该ScFv用于HER2阳性肿瘤的靶向基因治疗奠定了基础.AIM： To construct a fusion protein gene of human anti-HER2 ScFv/light chain constant region/truncated protamine （ScFv/Ck/tP） and analyze the binding activity of the expressed protein. METHODS： Two pairs of oligonucleotide primers were designed and used to amplify the SeFv and the Ck gene. After they were linked as ScFv/Ck, the synthesized tP coding sequence was added in the 3＇ terminus of it, then the fusion protein gene ScFv/Ck/tP was 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 antigen-binding activity of the ScFv/Ck/tP fusion protein was confirmed by cellular ELISA, and the DNA binding ability was confirmed by gel shift assay. RESULTS： Restriction endonuclease digestion and DNA sequencing proved that ScFv/ Ck/tP was correctly cloned into expression vector. SDS-PAGE and Western Blot analysis showed that ScFv/Ck/tP fusion protein was successfully expressed in E. coli BL21. Cellular ELISA confirmed that it had specific antigen binding activity; gel shift assay assured that it had DNA binding ability. CONCLUSION： The ScFv/Ck/tP fusion protein expressed in E. coli could specially bind with both HER2 antigen and DNA.

关 键 词：人源抗HER2单链抗体 轻链恒定区 鱼精蛋白 基因构建 活性鉴定 

分 类 号：Q78[生物学—分子生物学]