hHT/GFP融合基因表达载体的构建及在小鼠成纤维细胞中的表达

Construction of hHT/GFP Fusion Gene Expression Vector and Expression in Mouse Fibroblast

为探讨超急排斥反应(hyperacute rejection,HAR)及延迟性异种排斥反应(delayed xenograft rejec-tion,DXR)分子调控机制,进行-α1,2岩藻糖苷转移酶基因(hHT)与绿色荧光蛋白基因(green fluorescent protein,GFP)融合表达载体pEGFP-C1-hHT的构建及在小鼠成纤维细胞表达的研究。采用脂质体法转染小鼠成纤维细胞48 h后观察到GFP阳性细胞。同时对GFP阳性小鼠成纤维细胞进行PCR及Western blot,检测hHT基因的整合及表达情况。PCR结果表明hHT基因整合入小鼠基因组,Western blot检测到hHT基因的表达。研究结果表明,成功克隆hHT基因并构建了融合表达载体pEGFP-C1-hHT,可应用于进一步转基因研究。

To discuss the molecular mechanism of hyperacute rejection（HAR） and delayed xenograft rejection（DXR）,pEGFP-C1-hHT,the fusion expression vector of human alpha1,2-fucosyltransferase（hHT） and green fluorescent protein（GFP）,was constructed and was expressed in mouse fibroblast.The results of PCR proved hTH was cloned and the results of restrictive enzyme digestion analysis proved the expression vector pEGFP-C1-hHT was constructed.The positive signal of GFP was observed in mouse fibroblast into which pEGFP-C1-hHT was transferred.The fibroblast with positive signal of GFP was analyzed by PCR and western blot.The integration of hHT was proven by PCR.The expression of hHT was proven by western blot.These results showed that the fusion expression vector pEGFP-C1-hHT was constructed,and this vector could be used in future research work.