摘要
目的:构建、表达和鉴定抗转铁蛋白受体(TfR)单链抗体(scFv)-次级淋巴组织趋化因子(SLC)融合蛋白。方法:以pDsRed2-N1-CCL21为模板,设计引物,采用PCR扩增得到两端有EcoRⅠ、NotⅠ酶切位点的SLC基因。以EcoRⅠ、NotⅠ双酶切pET28a+scFv载体和PCR产物,连接后转化大肠杆菌BL21,IPTG诱导TfRscFv-SLC融合蛋白的表达。SDS-PAGE、Western blot分析TfR scFv-SLC的表达特性。FCM测定scFv-SLC与肿瘤细胞结合活性。结果:EcoRⅠ和NotⅠ双酶切鉴定可见约350bp的小片段,与SLC的理论值相符,DNA测序结果表明含有TfRscFv-Linker-SLC序列,表明pET28a+scFv-SLC融合基因表达载体构建成功。IPTG诱导产物经SDS-PAGE分析可见约41kD的条带,符合scFv-SLC理论值。FCM结果显示TfR scFv-SLC可与MCF-7、HepG2细胞结合,且结合的阳性率较TfRscFv有所提高。结论:成功构建与表达scFv-SLC融合蛋白,且能与MCF7、HepG2等细胞特异性结合。
Objective：To construct and express scFv-SLC against transferrin receptor and CCR7, and to identify its cell conjugation activity, nethods：PCR primers were designed according to the complementary sequences of gene SLC in the vector of pDsRed2-N1-CCL21, respectively, which contained inter-linker G4S and the restriction endonuclease EcoR Ⅰ and Not Ⅰ . SLC fragments was first amplified through PCR and then inserted into plasmid pEIE8a ＋ scFv, thereby producing a vector which could express a scFv-SLC-His tag（pTfR scFv-SLC）. To express scFv-SLC, E. coli. BL21 was cultured in LB broth and was induced by IPTG. The protein was analyzed by SDS-PAGE and Western blot. The cell conjugation activity of scFv-SLC was characterized by FCM. Results： It was demonstrated by digesting and sequencing results of pTfR scFv-SLC that the constructing was successful. Protein was analyzed by SDS-PAGE and Western blot, and the molecular weight was identical with the protein size of TfR scFv-SLC. The results of FCM proved that TfR scFv-SLC had the specificity of conjugating with TfR and CCR7 on the cell surface. Conclusion：TfR scFv-SLC against transferrin receptor and CCR7 have been successfully constructed and expressed. Expression products could specifically bind to TfR and CCR7 on the cell surface of MCF-7 and HepG2 cell.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2009年第9期832-834,843,共4页
Chinese Journal of Immunology
基金
"863"国家高新技术发展计划(2006AA02Z158)
教育部博士点专项资金(20060487024)
新教师基金课题(20070487103)
湖北省卫生厅重点科技攻关项目(JX3A02)资助
