AIF基因真核表达载体的构建及其在MGC-803中的表达

目的]检测胃癌标本中AIF基因是否存在碱基突变；构建AIF基因真核表达载体AIF—GFP；并检测其在胃腺癌细胞株MGC-803中的表达。方法]应用RT—PCR的方法分别从5例胃癌组织标本中提取总RNA，进一步扩增出全长的AIF基因；将其克隆到DMD19-T载体进行测序，并亚克隆至真核表达载体pEGFP—N2，重组载体AIF—GFP采用脂质体法瞬时转染MGC-803细胞，荧光显微镜观察AIF—GFP在细胞中的表达：结果]由5例胃癌组织标本提取的mRNA所扩增出的AIF基因的序列与GeneBank中AIF（AF100928）序列完全一致．胃癌组织中均未发现碱基突变；构建了真核表达载体AIF—GFP；并将其转染MGC-803进行表达。结论]胃癌中AIF基因未发生碱基突变；AIF-GFP真核表达载体，在MGC-803细胞中获得了表达，为进一步探讨AIF在胃癌诊治中的应用奠定了基础。

Creation of AIF Eukaryotic Expression Vector and Its Expression in MGC-803

Purpose] To analyze the gene mutation of AIF in gastric cancer samples and to creat the AIF eukaryotic expression vector AIF-GFP and to detect its expression gastric adenocarcinoma cell line MGC-803. Methods] Total RNA was extracted from tumor tissues of 5 cases with gastric cancer, and then the full-length AIF cDNA was obtained by RT-PCR. The products were cloned into pMD19-T vector, sequenced and then subcloned into eukaryotic expressing vector pEGFP-N2. The recombined vector AIF-GFP was transfected into MGC-803 ceils. The expression of AIF-GFP was observed by fluorescent microscope. Results ] The cDNA of AIF was sequenced and consistent with the sequence reported in Genebank （AF100928）. There is no mutation of AIF in the 5 cases. AIF eukaryotic expression vector AIF-GFP was successfully created and its expression in MGC-803 could be observed. Conclusions] There is no gene mutation of AIF in gastric cancer, and AIF eukaryotic expression vector AIF-GFP was expressed in MGC-803 cells, which provide basis for further study of AIF on diagnosis and treatment of gastric cancer.