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目的 :构建正义和反义人肝素酶与绿色荧光蛋白真核共表达载体 ,并分别转染人低转移肺癌细胞株 95C及高转移细胞株 95D .方法 :采用基因重组技术 ,用EcoRⅠ从pcD NA3 Hpa质粒上切下约 1 .7kb的人肝素酶全长cDNA片段 ,然后连入pIRES2 EGFP质粒的EcoRⅠ酶切位点上 ,经BamHⅠ酶切鉴定出正义和反义表达载体 ,并用脂质体法将肝素酶正义真核表达载体转入人低转移肺癌细胞株 95C、反义真核表达载体转入人高转移肺癌细胞株 95D ,G4 1 8筛选后 ,荧光倒置显微镜检测转染结果 .结果 :经BamHⅠ酶切后 ,正义质粒形成 5 .3+1 .7kb两条DNA片段 ,而反义重组质粒为 6 .5和 0 .5kb两条DNA片段 ,与理论计算值完全一致 ;转染后肺癌细胞倒置荧光显微下呈绿色荧光 .结论 :成功构建了人肝素酶的正、反义真核表达载体 ,并成功转染人低、高转移肺癌细胞株 ,为进一步研究正、反义肝素酶基因转染对肿瘤细胞的影响奠定了基础
OBJECTIVE: To construct eukaryotic co-expression vector of sense and antisense human heparanase and green fluorescent protein and to transfect human low metastatic lung cancer cell line 95C and high metastatic cell line 95D respectively.Methods: Using gene recombination technology, NA3 Hpa plasmid about 1.7 kb human heparanase full-length cDNA fragment was cut, and then connected to the pIRES2 EGFP plasmid EcoRI digestion site, identified by restriction endonuclease BamHI sense and antisense expression vector, and lipid Heparinase sense eukaryotic expression vector was transferred into human low metastatic lung cancer cell line 95C and the antisense eukaryotic expression vector was transfected into human highly metastatic lung cancer cell lines 95D and G418. The transfection results were detected by fluorescence inverted microscope Results: After digested with BamHⅠ, the two plasmids formed 5.3 ± 1.7kb two DNA fragments and the antisense recombinant plasmids were 6.5 and 0.55kb DNA fragments, which were completely consistent with the theoretical values. Infection of lung cancer cells under inverted fluorescence microscopy was green fluorescence.Conclusion: The positive and antisense eukaryotic expression vectors of human heparanase were successfully constructed and successfully transfected into human low and high metastatic lung cancer cell lines for further study of positive, Antisense heparanase gene transfection swollen The impact of tumor cells laid the foundation