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AIM:To construct and identify the recombinant vectorscarrying herpes simplex virus thymidine kinase (HSV-TK) andtumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2)genes expressed in gastric cardnoma cell line SGC7901.METHODS:The fragments of HSV-TK,internal ribosomeentry sites (IRES) and TNF-α or IL-2 genes were inserted ina TK-IRES-TNF-α or TK-IRES-IL-2 order into pEGFP-N_3 andpLXSN to generate the therapeutic vectors pEGFP-TT,pEGFP-TI, pL(TT)SN and pL(TI)SN respectively,which werestructurally confirmed by the digestion analysis of restrictionendonuclease.The former two plasmids were used for thetransient expression of recombinant proteins in the targetcells while pL(TT)SN and pL(TI)SN were transfected intoSGC7901 cells by lipofectamine for the stable expression ofobjective genes through G418 selection.The protein productsexpressed transiently and stably in SGC7901 cells by theconstructed vectors were confirmed by fluorescent microscopyand Western blot respectively.RESULTS:The inserted fragments in all constructed plasmidswere structurally confirmed to be consistent with that of thepublished data.In the transient expression,both pEGFP-TTand pEGFP-TI were shown expressed in nearly 50% of thetransfected SGC7901 cells.Similarly,the G418 selectedvectors PL(TT)SN and PL(TI)SN were confirmed to besuccessful in the stable expression of the objective proteinsin the target cells.CONCLUSION:The constructed recombinant vectors in thepresent study that can express the suicide gene TK incombination with cytokines genes may serve as the potentialtools to perform more effective investigations in future forthe gene therapy of gastric carcinoma.
AIM: To construct and identify the recombinant vectorscarrying herpes simplex virus thymidine kinase (HSV-TK) and tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2) genes expressed in gastric cardnoma cell line SGC7901.METHODS: The fragments of HSV-TK, internal ribosome sites (IRES) and TNF-α or IL-2 genes were inserted in TK-IRES-TNF-α or TK-IRES-IL-2 order into pEGFP-N_3 and pLXSN to generate the therapeutic vectors pEGFP Which werestructurally confirmed by the digestion analysis of restriction endonuclease. The former two plasmids were used for the transient expression of recombinant proteins in the target cells while pL (TT) -TT, pEGFP-TI, pL (TT) SN and pL SN and pL (TI) SN were each transfected into SGC7901 cells by lipofectamine for the stable expression of ajective genes through G418 selection.The protein product was expressed in transiently and stably in SGC7901 cells by theconstructed vectors were confirmed by fluorescent microscopy and Western blot respectively.RESULTS: The inserted fragments in all constructed plasmidswere structurally confirmed to be consistent with that of the published data. In the transient expression, both pEGFP-TTand pEGFP-TI were shown in nearly 50% of the transfected SGC7901 cells. Similarly, the G418 selectedvectors PL ) SN and PL (TI) SN were confirmed to be successful in the stable expression of the objective proteins in the target cells. CONCLUSION: The constructed recombinant vectors in the present study that can express the suicide gene TK incombination with cytokines genes may serve as the potential tools to perform more effective investigations in future forthe gene therapy of gastric carcinoma.