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目的:构建靶向结肠癌细胞的人端粒酶逆转录酶HTERT启动子调控的NK4基因重组腺病毒并对该表达体系进行鉴定。方法:根据GenBank中的HTERT启动子(HTERTp)序列设计引物,以人胚肾293细胞提取基因组DNA为模板,PCR扩增HTERTp,并将HTERTp序列克隆入pDC312质粒中,构建成HTERTp调控的腺病毒质粒pSG-HTERTp;NK4 cDNA酶切并回收克隆至pSG-HTERTp载体中,构建成pSG-HTERTp/pA-NK4穿梭质粒,脂质体法在293细胞中包装重组腺病毒;噬斑分析法筛选单克隆重组腺病毒;RT-PCR检测NK4基因的转录;Western Blot检测NK4蛋白的表达。结果:成功构建出成功构建出HTERT启动子调控的NK4基因重组腺病毒,滴度为4.5×10~9空斑形成单位(pfu)/mL,且PCR扩增及westernbolt电泳均呈阳性表达。结论:成功构建出HTERT启动子调控的NK4基因重组腺病毒,为进一步研究NK4基因的作用机制打下实验基础。
OBJECTIVE: To construct a recombinant adenovirus of NK4 gene regulated by HTERT promoter of human telomerase reverse transcriptase targeted to colon cancer cells and identify the expression system. Methods: According to the HTERT promoter sequence in GenBank, primers were designed. Genomic DNA was extracted from human embryonic kidney 293 cells as a template to amplify HTERTp by PCR. The HTERTp sequence was cloned into pDC312 plasmid to construct HTERTp-regulated adenovirus Plasmid pSG-HTERTp; NK4 cDNA was digested and cloned into pSG-HTERTp vector to construct pSG-HTERTp / pA-NK4 shuttle plasmid. The recombinant adenovirus was packaged in 293 cells by lipofectamine. The recombinant adenovirus was cloned. The transcription of NK4 gene was detected by RT-PCR. The expression of NK4 protein was detected by Western Blot. Results: The recombinant adenovirus of NK4 gene successfully constructed by HTERT promoter was successfully constructed with a titer of 4.5 × 10 ~ 9 pfu / mL and was positive for PCR amplification and westernbolt electrophoresis. Conclusion: The recombinant adenovirus of NK4 gene regulated by HTERT promoter was successfully constructed, which laid the experimental foundation for further study on the mechanism of NK4 gene.