摘要
目的 :为进行TNFR的靶向基因治疗研究打基础。方法 :将人TNFR55和TNFR75基因序列分别与KDR启动子 (KDRp)及灭活自身启动子的逆转录病毒载体RV D2 99重组 ,构建RV D2 99 KDRp TNFR55及RV D2 99 KDRp TNFR75逆转录病毒载体 ,分别转染包装细胞PA31 7,获得稳定的产病毒细胞系。 结果 :获得的TN FR55和TNFR75产毒细胞系病毒滴度分别为 1× 1 0 5CFU ml和 2× 1 0 5CFU ml。感染RV D2 99 KDRP TNFR55病毒的ECV30 4细胞与TNF孵育后的培养上清 ,对L92 9细胞的细胞毒活性比相同条件下的NIH3T3细胞低约2 6倍 ,而感染RV TNFR55病毒的ECV 30 4与相同条件下的NIH3T3细胞无明显差异。结论 :建立了TNFR55和TNFR75逆转录病毒高产毒细胞系 ,构建的RV D2 99 KDRP TNFR55和RV D2 99 KDRP
Objective:To make the basis for the TNFR target gene therapy Methods:In order to construct RV D 299 KDRp TNFR55 and RV D 299 KDRp TNFR75 retrovirus vector,the cDNAs coding TNFR55 and TNFR75 were separately recombined with KDR promoter(KDRp)and Retrovirus Vector RV D 299 which could make the promoter of itself inactive These two kinds of recombinants were transfected into packaging cell PA317 separately, screened by G418,the stable cell strain producing toxin was obtained Results: The titer of virus obtained from TNFR55 and TNFR75 infected toxin producing cell strain were 1×10 5 CFU/ml and 2×10 5 CFU/ml separately After incubated with TNF, the cytotoxicity of culture supernant of ECV304 cell which was infected with RV D299 KDRP TNFR55 to L929 cell was about 2 6 folds lower than that in NIH3T3 in the same condition, but the cytotoxicity of culture supernant of ECV304 cell which was infected with RV TNFR55 to L929 cell was no significant difference from the NIH3T3 cell in the same condition Conclusion: the TNFR55 and TNF75 retrovirus high producing poison cell strain was constructed The constructed RV D 299 KDR P TNFR55 and RV D 299 KDR P TNFR75 recombinants could mediate the target expression of TNFR gene in vascular endotheliocyte
出处
《军医进修学院学报》
CAS
2002年第3期220-222,共3页
Academic Journal of Pla Postgraduate Medical School
