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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 the pmRNAIRES-hKDR recombinant plasmid vector using genetic engineering technology and lay a foundation for gene therapy research of tumor. Methods The pDC520hKDR conserved in our laboratory was digested with XbaⅠand NcoⅠ. PmRNAIRES-Luc was also digested with Xba and NcoⅠ. The pmRNAIRES-hKDR recombinant plasmid was constructed by molecular biology techniques and identified by PCR and restriction enzyme digestion Lipofectamine was transfected into Hepall-6 cells, which were screened by G418 and detected by ELISA and Western blot. Results The recombinant plasmid contained pmRNAIRES-hKDR gene. After transfected into Hepall-6 cells, the expressed product secreted into the tumor cells. The secreted extracellular products were proved to bind to specific hKDR antibodies by Western blot. Conclusion The recombinant plasmid containing eukaryotic expression vector pmRNAIRES-hKDR was successfully constructed, which is helpful to further study its anti-tumor effect.