摘要
用HindⅢ将HIV-1Tat101蛋白编码基因从pEV质粒中切出,BamHI、NotⅠ将绿色荧光蛋白(GFP)编码基因从表达质粒pcDNA3 1+/GFP中切出,分别插入到质粒LZRSpBMN-Z中,构建成重组反转录病毒表达质粒LZRS-Tat101和LZRS-GFP。采用磷酸钙转染法将两重组质粒转染到含反转录病毒env、gal和pol编码基因的包装细胞Phoenix(ΦNX)中,嘌呤霉素筛选获得稳定细胞系。分别收集稳定细胞系分泌的病毒上清,并感染体外培养的原发性渗出性淋巴瘤(PEL)BCBL-1细胞。收集LZRS-GFP重组病毒感染的BCBL-1细胞进行流式细胞计数,检测GFP表达水平。收集LZRS-Tat101重组病毒感染的BCBL-1细胞,提取蛋白作Westernblot,检测Tat蛋白表达状况;取细胞总RNA作Northernblot和定量PCR,检查HHV-8次要衣壳蛋白ORF26mRNA转录水平。重组LZRS-Tat101病毒进一步感染HL3T1细胞(HeLa细胞包含HIV-1-LTR/CAT报告基因),收集感染细胞提取蛋白,检测CAT活性,评价Tat生物学功能。PCR扩增HHV-8复制和转录激活蛋白Rta启动子区上游序列,并克隆至pGL-3载体中,构建Rta启动子+虫荧光素酶(Luciferase)报告基因重组质粒。此重组质粒进一步电转染预先感染了LZRS-Tat101病毒的BC-3细胞,TPA刺激后收集细胞,检测Luciferase活性。结果显示:①重组反转录病毒感染BCBL-1细胞,一次感染?
Human immunodeficiency vi rus type 1(HIV-1)Tat_(101) encoding gene and green fluorescence protein(GFP)enc oding gene were separately recover ed from pEV plasmid by HindⅢ diges tion and from pcDNA3.1+/GFP by Bam HI/NotⅠ digestion and then were se parately cloned into the expressio n plasmid LZRSpBMN-Z to construct the recombinant retroviral express ion plasmids named LZRS-Tat_(101) and LZRS-GFP,respectively.Using calciu m phosphate,constructs were then s eparately transfected into packagi ng cell line Phoenix(ΦNX)which con tained env and gal encoding for st ructural proteins while pol encodi ng for 3 enzymes(reverse transcrip tase,protease and integrase)essent ial for retroviral integration and replication.Stable transfected cel l lines were obtained by using pur omycin to screen for more than 7 d ays.Supernatants containing recomb inant virus collected from stably transfected cells were employed to infect BCBL-1 cell respectively an d the expressed Tat in infected BC BL-1 cell was tested by Western bl ot.The expressed GFP in infected B CBL-1 cell was detected by using f low cytometry to evaluate the infe ction efficiency of this retroviru s.Meantime,the HL3T1 cell which ac tually is HeLa cell line containin g a HIV-1-LTR/CAT reporter constru ct was infected with supernatant c ontaining Tat virus and then teste d for CAT activity.Northern blot a nd quantitative PCR(real-time PCR) were carried out to detect mRNA le vel of ORF26(minor capsid protein) of human herpesvirus 8(HHV-8)from T at virus infected BCBL-1.The resul ts were extended and confirmed usi ng a luciferase reporter construct driven by the HHV-8 Rta(Rta stands for replication and transcription activator,also named ORF50)promote r,the first promoter activated dur ing HHV-8 replication.The results were shown as follows:①56% of the cells expressed GFP when BCBL-1 ce ll was infected one time with reco mbinant retrovirus LZRS-GFP;②Tat c ould be detected in BCBL-1 cell in fected with Tat virus and was suff icient to activate HIV-1 LRT promo ter in HL3T1,resulting in the down stream high expression of CAT;③Tat could not induce HHV-8 Rta promoter activity;④Tat expressed in BCBL-1 failed to demonstrate ORF26 induct ion.According to these data,it is suggested that Tat itself is not s ufficient to activate lytic cycle replication of HHV-8.
出处
《病毒学报》
CAS
CSCD
北大核心
2003年第4期306-312,共7页
Chinese Journal of Virology
基金
国家自然科学基金项目(30100160和30271179)
