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目的:构建携带单纯疱疹病毒脱氧胸腺嘧啶激酶基因(herpes simplex virus thymidine kinase,HSV-TK)的逆转录病毒,用于宫颈癌的治疗研究。方法:用限制性内切酶从质粒pcDNA3.1/HA-myc-His(-)Z-TK切下HSV-1TK cDNA序列,亚克隆入逆转录病毒载体pLXSN得到重组质粒pLXSN-TK,鉴定正确的阳性重组质粒经PA317细胞包装,G418筛选,在NIH3T3细胞进行病毒滴度测定。然后用病毒感染人宫颈癌细胞HeLa。PCR、RT-PCR和Western blotting方法检测HSV-1TK基因在HeLa中的整合和表达情况。结果:重组质粒pLXSN-TK经PA317细胞包装后收获病毒上清,感染HeLa细胞,检测发现HSV-1TK基因整合到细胞基因组DNA中,并且能有效的转录和翻译。结论:成功构建了逆转录病毒pLXSN-TK,该病毒能有效感染HeLa细胞,并使携带的治疗基因HSV-1TK在细胞中表达,为今后HSV-1TK基因治疗宫颈癌的研究奠定基础。
Objective: To construct a retrovirus carrying herpes simplex virus thymidine kinase (HSV-TK) for the treatment of cervical cancer. Methods: HSV-1TK cDNA was excised from the plasmid pcDNA3.1 / HA-myc-His (-) Z-TK by restriction enzyme and subcloned into the retroviral vector pLXSN to obtain the recombinant plasmid pLXSN-TK, which was identified correctly Of the positive recombinant plasmid PA317 cell packaging, G418 selection, NIH3T3 cells in the virus titer determination. The virus is then used to infect human cervical carcinoma HeLa cells. The integration and expression of HSV-1TK gene in HeLa were detected by PCR, RT-PCR and Western blotting. Results: The recombinant plasmids pLXSN-TK were packaged in PA317 cells and harvested. The virus supernatant was harvested and infected into HeLa cells. The HSV-1TK gene was integrated into the genomic DNA of the cells and could be efficiently transcribed and translated. CONCLUSION: The recombinant retroviral vector pLXSN-TK was successfully constructed, which can effectively infect HeLa cells and express HSV-1TK in the cells, which will lay the foundation for future research on HSV-1TK gene therapy for cervical cancer.