pmRNA IRES-hKDR(Ig1-3)重组质粒的构建、表达及鉴定  

Construction,Expression and Identification of Recombinant Plasmid with pmRNA IRES-hKDR(Ig1-3)

作　　者：孙林英[1] 刘畅[2] 胡锋涛[3] 刘秀丽[4] 邵晓婷[4] 刘端祺[2] 谭晓华[4] 

机构地区：[1]泰山医学院医学检验系,山东泰安271016 [2]北京军区总医院肿瘤诊疗中心,北京100700 [3]滕州市级索中心卫生院,山东滕州277500 [4]北京军区总医院血液科,北京100700

出　　处：《华北国防医药》2008年第4期5-7,F0002,共4页Medical Journal of Beijing Military Region

基　　金：国家自然科学基金资助项目(30371627);北京市自然科学基金资助项目(7042062)

摘　　要：目的:利用基因工程技术构建pmRNA IRES-hKDR(Ig1-3)重组质粒,为其应用于肿瘤的基因治疗奠定基础。方法:以本实验室构建保存的pcDNA3.1 hKDR为模板,PCR扩增hKDR(Ig1-3)cDNA片段后用Nco I和Xho I酶切后插入pmRNA IRES多克隆位点的相应位点中,经PCR、酶切和测序鉴定其序列正确性,然后用试剂盒体外转录出相应的mRNA,用脂质体转染COS-7细胞,经G418筛选后通过免疫组化和Western blot检测该融合蛋白。结果:PCR、酶切和测序证实pmRNA IRES-hKDR(Ig1-3)构建成功,体外转录出相应的mRNA,免疫组化和Western blot检测出其蛋白表达。结论:成功构建含pmRNA IRES-hKDR(Ig1-3)的真核表达重组质粒,有助于进一步研究其抗肿瘤作用。Objective：To construct recombinant plasmid pmRNA IRES- hKDR （Ig1-3）and lay a primary foundation for further study of hKDR（Igl-3） gene therapy of tumors. Methods： pmRNA IRES-hKDR （Igl-3）was obtained from pcDNA3, l hKDR and pmRNA IRES-Luc through molecular biology technology,pcDNA3.1 hKDR and pmRNA IRES-Luc were reserved by our laboratory. After identification of PCR, restriction enzyme digesting and DNA sequencing,mRNA was transcripted in vitro and transfected into Cos-7 cell line via lipsome. The transfeeted cells, cos-7 ,were screened by C418. And then immunohistochemistry technique （IHCT） and Western blot were performed to identify the fusion protein. Results：The construction was verified by PCR ,restriction enzyme digesting and DNA sequencing, pmRNA was transeripted in vitro and protein expression by immunohistochemistry technique （IHCT）and western blotting was detected. Conclusion： An eukaryotic expression recombinant plasmid pmRNA IRES-hKDR （Ig1-3）can be constructed successfully and provide the possibility of further research on its anti-tumor effect.

关 键 词：受体 血管内皮生长因子 质粒 基因表达调控 

分 类 号：R349.8[医药卫生—基础医学]