615小鼠MHCⅠcDNA重组逆转录病毒基因治疗载体的构建和包装
Reconstruction and Packeting of the MHC I cDNA of 615 Mice Recombinant Retrovirus Vectors
作者：龚浩,李龙江,温玉明,王昌美,陈俊杰,彭文珍
Author：Gong Hao, Li Longjiang, Wen Yuming, et al
收稿日期：2002-12-25 年卷（期）页码：2002,20(06):404-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：小鼠,MHCⅠ,转染,PA317细胞,
Key words：mouse,MHC I,transfection,PA317 cell,
基金项目：国家自然科学基金资助项目(编号 39700162)
中文摘要
目的:制备615小鼠MHCⅠ目的基因(H-2Kk)载体,获得高表达MHCⅠ分子功能的单克隆细胞株。方法:将615小鼠MHCⅠ(H-2Kk)cDNA定向连接至PLXSN逆转录病毒质粒,转染E.coli JM109,限制性酶切分析法筛选出重组质粒PLXSN-H-2Kk,继而转染PA317细胞,经G418筛选获得抗性PA317单克隆细胞株,并转染NIH3T3细胞,依抗性NIH3T3细胞克隆数目,鉴定病毒液的滴度和PA317单克隆细胞株。结果:转染成功的病毒液滴度位于1.6×104～1.1×105 CFU/ml之间,PA317单克隆细胞株成片生长良好,并高表达H-2Kk产物。结论:H-2Kk cDNA重组逆转录病毒质粒的成功构建、包装和滴度鉴定为在615小鼠上进行恶性肿瘤的MHC Ⅰ转基因治疗奠定了物质基础。
英文摘要
Objective: The aims of this study were to prepare the vector of MHCI functional gene ( H-2Kk) of 615 mice, and to get the monoclone cell strains with MHC I molecule function.Methods: The MHC I (H-2Kk) cDNA of 615 mouse was inserted into PLX-SN retrovirus plasmid, and E. coli JM109 was transformed by the ligated product. The recombinant plasmid PLXSN-H-2K was obtained by restriction enzyme analysis. The PAS 17 packet cells were transfected by the recombinant plasmid. The transfected PA317 cells were screened under the G418 selective stress. The G418 resistant monoclone cell strains were picked up and employed to prepare the retrovirus solutions. The NIH3T3 cells were transfected by the retrovirus solutions in different dilutions. The titres of the retrovirus solutions were determined by counting the G418 resistant NIH3T3 cell clones, and the G418 resistant mono-clone PAS 17 cell strain which could produce high litre retrovirus solution was selected.Results: The titres of the retrovirus solutions with successful transfection were 1.6 x 104 to 1.1 x 105 colony forming units (CFU) per ml. The PAS 17 monoclone cell strains grew well with the patches, and produced many products of H-2Kk. Conclusion: The reconstruction and packeting of the recombinant retrovirus plasmid PLXSN-H-2K cDNA could be available in further study on MHC I transgenic therapy for malignant tumor on 615 mice.
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