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目的:构建含HIV-1 Tat基因重组反转录病毒表达载体及评价表达Tat蛋白的功能。方法:使用Hind Ⅲ将HIV-1 Tat_(101)蛋白编码基因从pEV质粒中切出,插入到表达质粒LZRSpBMN-Z中,构建成重组反转录病毒表达质粒LZRS-Tat_(101)。采用磷酸钙转染法将重组质粒LZRS-Tat_(101)转染进含反转录病毒env、gal和pol编码基因的包装细胞phoenix(ΦNX)中,嘌吟霉素筛选获得稳定细胞系。免疫组化(IHC)染色检查Tat在临时转染和稳定转染ΦNX细胞中的表达水平。收集临时转染和稳定转染包装细胞分泌的病毒上清,并分别感染293细胞,Westemblot检测Tat在293中表达。与此同时,收集感染293细胞培养上清,加入到HL3T1细胞(HE-La-HIV-1-LTR/CAT报告基因)中,共培养72 h后收集细胞,提取蛋白作CAT活性检测。结果:①含Tat_(101)基因重组反转录病毒表达质粒转染包装细胞后,Tat在临时转染ΦNX中表达水平显著高于在稳定转染中的水平;②临时转染病感染293细胞,Tat在感染细胞中得到了表达,且分泌至上清中的Tat蛋白能够激活靶细胞HL3T1中HIV-1的LTR启动子,使得其下游的CAT基因得到表达。结论:重组LZRS-Tat_(101)反转录病毒能够在其感染靶细胞中表达Tat蛋白,且表达蛋白具有转录激活功能。
OBJECTIVE: To construct recombinant retroviral vector containing HIV-1 Tat gene and to evaluate the expression of Tat protein. Methods: The gene encoding HIV-1 Tat 101 protein was excised from pEV plasmid using Hind III and inserted into expression vector LZRSpBMN-Z to construct recombinant retrovirus expression plasmid LZRS-Tat_ (101). The recombinant plasmid LZRS-Tat (101) was transfected into the packaging cell phoenix (ΦNX) containing the retroviral env, gal and pol genes by calcium phosphate transfection method and screened by puromycin to obtain a stable cell line. Immunohistochemistry (IHC) staining was used to examine the expression level of Tat in transiently transfected and stably transfected ΦNX cells. The virus supernatants collected from transiently transfected and stably transfected packaging cells were collected and 293 cells were infected respectively. The expression of Tat was detected in 293 by Westemblot. At the same time, the supernatant of infected 293 cells was collected and added to the HL3T1 cells (HE-La-HIV-1-LTR / CAT reporter gene). After co-cultured for 72 hours, the cells were harvested and the proteins extracted for CAT activity assay. Results: (1) Tat transfected packaging cells containing Tat (101) gene recombinant retrovirus showed that the expression level of Tat in transiently transfected ΦNX was significantly higher than that in stable transfected cells. ② The transient transfected cells infected 293 cells , Tat was expressed in infected cells and the Tat protein secreted into the supernatant was able to activate the LTR promoter of HIV-1 in target cell HL3T1 so that the downstream CAT gene was expressed. CONCLUSION: The recombinant LZRS-Tat (101) retrovirus can express Tat protein in the infected target cells, and the expressed protein has transcriptional activation.