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

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-KDR_P-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-KDR_P-TNFR55 and RV-D₂₉₉-KDR_P-TNFR75 recombinants could mediate the target expression of TNFR gene in vascular endotheliocyte.