615小鼠MHCⅠcDNA重组逆转录病毒基因治疗载体的构建和包装

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.