pmRNA IRES-hKDR重组质粒的构建、表达及鉴定

Construction,expression and examinations of recombiant plasmid with pmRNA IRES-hKDR

目的利用基因工程技术构建pmRNAIRES-hKDR重组质粒载体,为肿瘤的基因治疗研究奠定基础。方法利用本实验室保存的pDC520hKDR用XbaⅠ和NcoⅠ顺序酶切,pmRNAIRES-Luc亦用XbⅠ和NcoⅠ顺序酶切,通过分子生物学技术构建了pmRNAIRES-hKDR重组质粒,用PCR、酶切鉴定,然后用脂质体转染Hepall-6细胞,用G418筛选后通过ELISA和Western blot检测该融合蛋白。结果重组质粒中含有pmRNAIRES-hKDR基因,转染Hepall-6细胞后,其表达产物能分泌到肿瘤细胞外,分泌到细胞外的产物用Western blot检测证明能与特异性hKDR抗体结合。结论成功构建含pmRNAIRES-hKDR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 recobinant pmRNA IRES-hKDR and lay a primary foundation for further study of gene therapy for tumors. Methods pmRNA IRES-hKDR was obtained from pDC520 hkDR and pmRNA IRES-Luc which was reserved by our laboratory through molecular biology technology and was transfected into Hepall-6 via lipsome after identification of PCR,restriction enzyme digesting and DNA ELISA and Western blot. Resuits The recombiant plasmid had pmRNA IRES-hKD gene which was expressed in Hepall-6//G418 in vitro and the expressed protein was secreted out of the cells could response with specific hKDR antibody, which was identified by Western blot. Conclusion An eukaryotic expression recombiant plasmid pmRNA IRES-hKDR can be constructed successfully which provides the possibility of further researches on its anti-tumor effect.