KDR启动子驱动的TNFR逆转录病毒载体构建及其靶向表达

The construction of KDR promoter drive TNFR retrovirus vector and its target expression

  • 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-D299重组,构建RV-D299-KDRp-TNFR55及RV-D299-KDRp-TNFR75逆转录病毒载体,分别转染包装细胞PA317,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× 105CFU/ml和2×105CFU/ml。感染RV-D299-KDRP-TNFR55病毒的ECV304细胞与TNF孵育后的培养上清,对L929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-304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-D299-KDRP-TNFR55和RV-D299-KDRP-TNFR75重组病毒载体可以介导TNFR在血管内皮细胞中靶向表达。

     

    Abstract: Objective: To make the basis for the TNFR target gene therapy. Methods: In order to construct RV-D299-KDRpTNFR55and RV-D299-KDRp-TNFR75 retrovirus vector,the cDNAs coding TNFR55 and TNFR75 were separately recombined with KDR promoter(KDRp)and Retrovirus Vector RV-D299which could make the promoter of itself inactive.These two kinds of recombinants were transfected into packaging cell PA317separately,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 1×105 CFU/ml and 2×105CFU/ml separately.After incubated with TNF,the cytotoxicity of culture supernant of ECV304 cell which was infected with RV-D299-KDRP-TNFR55 to L929cell was about 2.6folds 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 TNFR55and TNF75retrovirus high producing poison cell strain was constructed.The constructed RV-D299-KDRP-TNFR55 and RV-D299-KDRP-TNFR75 recombinants could mediate the target expression of TNFR gene in vascular endotheliocyte.

     

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