表达mFasL的新型双顺反子逆转录病毒基因转移体系的建立  

Establishment of Gene Transfer System by a New Retroviral Vector Containing an Internal Ribosome Entry Site to Link Two Cistons and Expressing Mouse Fas Ligand

作　　者：刘凌波[1] 邹萍[1] 徐之良[2] 王良利[1] 胡中波[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院血液病学研究所,武汉430022 [2]武汉大学医学院附属人民医院儿科,武汉430060

出　　处：《华中科技大学学报（医学版）》2002年第2期136-139,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目 (No. 39770 76 7)

摘　　要：为建立表达小鼠 Fas配体 (m Fas L)的内部核糖体进入位点 (IRES)串联的双顺反子新型逆转录病毒基因转移体系 ,用基因重组技术将 m Fas L c DNA基因构建到 IRES双顺反子逆转录病毒载体 (PL XIN)中 ,脂质体法将此重组质粒 PL FIN和空载体 PL XIN分别转染包装细胞 (PA317) ,经 G418筛选出抗性包装细胞克隆 ,基因组 DNA PCR测定基因整合情况。并将抗性 PA317克隆培养上清感染小鼠成纤维细胞系 (NIH3T3) ,筛选出高滴度的 PA317细胞系 ;用流式细胞术测定筛选的 NIH3T3细胞目的基因表达状况 ;以抗性 NIH3T3和 Fas+Yac- 1细胞共培养法检测目的基因的生物学活性。筛选出 12个 PL FIN转染的 PA317细胞克隆中 ,最高产毒效价为 8.5× 10 5CFU(colony- form ing- unit) ;经此上清感染、筛选出的 NIH3T3细胞 ,高表达 m Fas L (阳性率高于对照组 5 2 .5 4% ) ;且能显著诱导 Fas+Yac- 1细胞凋亡 (凋亡率高于对照组 49% )。说明成功建立了表达 m Fas LTo establish gene transfer system of a new retroviral vector (PLXIN) containing an internal ribosome entry site to link two cistrons and expressing mouseFas ligand, FasL cDNA was recombined with PLXIN by gene recombination technology, the recombinant plasmid (PLFIN) and PLXIN were separately transferred into retrovirus packing cell line PA317 by lipofectamine 2000, and the resistant clones were selected by G418 selective medium. Integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transferred by the culture supernatants from PA317 carrying the mouse Fas ligand cDNA gene, and were selected by G418 selective medium, so as to get the PA317 clone capable of producing the supernatants of higher virus titers. The expression level of mFasL protein on NIH3T3 cell membrane was assayed by flow cytometer (FCM). The biological activity of mFasL expressed on NIH3T3 cell membrane was detected by the co-cultivation method of resistant NIH3T3 with Fas +Yac-1 cells. The recombinant plasmid PLFIN was successfully coustructed. The PLFIN-transferred PA317 cells clone, the culture supernatants of which had the highest titers of 8.5×10 5CFU/ml among 12 resistant cell clones, was discovered. The mFasL positive rate of the NIH3T3 cells transferred by the culture supernatants was 52.54 % higher than in the control group. The apoptosis rate of Fas +Yac-1 cells induced with the NIH3T3 cells was 49 % higher than control group. It was concluded that the gene transfer system of a new retroviral vector containing an internal ribosome entry site to link two cistrons and expressing mouse Fas ligand was established.

关 键 词：基因转移 细胞凋亡 mFasL 新型双顺反子逆转录病毒 

分 类 号：Q78[生物学—分子生物学]