携带HSV-1TK基因的逆转录病毒载体构建及在HeLa细胞中的表达

Construction of recombinant retroviral vector carrying herpes simplex virus thymidine kinase-1 gene and identification of its expression in HeLa cells

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 the recombinant retrovirus carrying herpes simplex virus thymidine kinase-1（HSV-1TK） gene and to observe the expression of HSV-1TK in cervical carcinoma cell line HeLa. Methods：The HSV-1TK cDNA was digested by restriction endonuclease from plasmid pcDNA3.1/HA-myc-His（-） Z-TK and then directionally inserted into retroviral vector pLXSN to construct recombinant plasmid pLXSN-TK. The pLXSN-TK was transferred into retroviral producer cell line PA317 for packaging retrovirus and then the resultant retrovirus was applied to infected the HeLa cells. The integration of HSV-1TK gene into the HeLa cells genome was assayed by PCR. The expression of HSV-1TK gene was detected by RT-PCR and Western blot. Results：The HSV-1TK gene was integrated into the genome of HeLa cells shown by results of PCR after retroviral infection. The expression of HSV-1TK gene was confirmed by RT-PCR and Western blotting. Conclusion：The recombinant retrovirus carrying HSV-1TK gene was constructed successfully and may be a potentially effective strategy for the treatment of cervical cancer.