目的 包装出携5拷贝缺氧反应元件(5HRE)增强子和癌胚抗原启动子(CEAp)联合调控的超抗原中毒性休克综合征毒素-1 (TSST-1)基因的逆转录病毒,并建立稳定表达TSST-1的人结肠癌Lovo细胞株.方法 应用已构建好的逆转录病毒载体pLEGFP N1 5HRE-CEAp TSST-1 linker-CD80TM,与辅助质粒pMB-MLV、pMB-G共转染入293T细胞,LaSRT法检测病毒滴度.包装出的逆转录病毒感染CEA阳性的人结肠癌细胞株Lovo和CEA阴性的人宫颈癌细胞株Hela.应用RT-PCR和免疫组化方法鉴定TSST-1的表达.结果 获得重组逆转录病毒滴度约为1×106 CFU/mL.RT-PCR及免疫组化证实经病毒感染的Lovo细胞在mRNA和蛋白水平均有TSST-1表达,缺氧环境中的mRNA表达量更高,且细胞传代20次后依然有TSST-lmRNA表达；经病毒感染的Hela细胞在常氧或缺氧状态下均无TSST-1mRNA及蛋白表达.结论 包装出较高滴度重组逆转录病毒,并能靶向性在CEA阳性肿瘤内稳定表达TSST-1,为后续检验TSST-1对肿瘤的杀伤效应奠定了基础.%Objective To package a recombinant retrovirus carrying the superantigen of toxic shock syndrome toxin-1 (TSST-1) gene, which is regulated synergistically by 5 copies of hypoxia-responsive element (5HRE) and promoter of carcino-embryonic antigen (CEAp) and identify TSST-1's expression in carcino-embryonic antigen (CEA)-positive human colon carcinoma cell line Lovo. Methods The recombinant retrovirus vector was constructed with the transmembrane superantigen gene of 5HRE-CEAp- regulated TSST-l-linker-CD80TM, and was co-transfected into packaging cell 293T with helper vector pMB-MLV and pMB-G. LaSRT method was employed to examine the titers of retrovirus. CEA-positive human colon carcinoma cell line Lovo and CEA-negative human cervical carcinoma cell line Hela were infected with recombinant retrovirus respectively. RT-PCR and immunohistochemistry were employed to examine the expression of TSST-1. Results The recombinant retrovirus was harvested and the titer was 1 X 106 CFU/mL. The expression of TSST-1 mRNA and protein in infected Lovo cells under hypoxia condition was significantly higher than that under normoxia condition as confirmed by RT-PCR and immunocytochemistry. It was still expressed after passaged 20 of the infected Lovo cells. Infected Hela cells did not express TSST-1 mRNA or protein under either hypoxia condition or normoxia condition. Conclusion The titer of recombinant retrovirus is enough to infect target cells, and could target and stably express TSST-1 in CEA-positive human colon carcinoma cell. All this provides a foundation for proving TSST-1's effect in killing CEA-positive tumor cells in follow-up tests.
展开▼
