pCMV-HSF2-FLAG重组质粒的构建及其在Caco-2细胞中的表达

Construction of a eukaryotic expression plasmid encoding the human HSF2 gene and its expression in Caco-2 cells

目的:构建人HSF2 pCMV-Myc真核表达载体,研究其在人结肠上皮肿瘤细胞Caco-2内的表达和定位.方法:以HSF2 Human cDNA ORF clone为模板,PCR法扩增出全长HSF2编码系列,用EcoRⅠ和KpnⅠ对HSF2 PCR纯化产物双酶切后,采用基因重组技术将其插入至pCMV-Myc载体中.DNA测序正确后,脂质体法将重组质粒转染到Caco-2细胞内,提取细胞总RNA逆转录为cDNA后进行普通PCR检测HSF2 mRNA转录水平,提取细胞蛋白进行Western blot检测HSF2蛋白质水平及融合蛋白表达.采用激光共聚焦显微镜观察HSF2及融合蛋白在Caco-2内的表达和定位.结果:将人全长HSF2编码序列克隆到真核表达载体pCMV-Myc中,酶切鉴定片段为1557bp,重组质粒测序正确.转染重组质粒后,PCR显示HSF2在mRNA水平较未转染组和空载组升高;Western blot检测到融合蛋白正确表达,分子量约为70kDa.与未转染组和空载组相比,重组质粒组HSF2蛋白质水平明显升高.激光共聚焦显微镜下观察到HSF2和融合蛋白定位一致,主要分布于Caco-2细胞质中,少量聚集在细胞核膜.结论:成功构建了人全长HSF2编码序列的pCMV-HSF2-FLAG真核表达载体,并在Caco-2细胞中成功表达,为进一步研究HS F2在溃疡性结肠炎中的作用奠定了良好的基础.

AIM：To construct a eukaryotic expression plasmid encoding the human heat shock factor 2 （HSF2） gene and to examine its expression and localization in Caco-2 cells, a human colon adenocarcinoma cell line using FLAG tag as a reporter. METHODS：The coding sequence of the HSF2 gene was amplified by PCR using human HSF2cDNA as the template and subcloned into pCMV-Myc vector after digestion with EcoR I and Knp I. After the identity of recombinant plasmid was verified by direct sequencing, the plasmid was transfected into Caco-2 cells using Lipofectamine. Total RNA was extracted, reverse transcribed into cDNA, and tested by PCR. The expression of HSF2 and the recombinant fusion protein in Caco-2 cells was detected by Western blot. The expression and localization of HSF2 and the recombinant fusion protein in Caco-2 cells were observed by laser scanning confocal microscopy. RESULTS：The coding sequence of the HSF2 gene was successfully inserted into the pCMV-Myc vector. Restriction enzyme digestion analysis showed that the length of the insert was 1 557 bp, matching the expected size. The mRNA level of HSF2 in cells transfected with the recombinant plasmid was higher than those in non-transfected cells and cells transfected with empty vector. The expression of recombinant HSF2-FLAG fusion protein, which had a molecular weight of 70 kDa, was detected by Western blot. The expression of HSF2 in cells transfected with the recombinant plasmid increased dramatically in comparison with matched groups. The HSF2 and recombinant HSF2-FLAG protein were localized predominantly to the cytoplasm but partially aggregated around the nuclear envelope in Caco-2 cells. CONCLUSION：The recombinant plasmid pCMV-HSF2-FLAG has been successfully constructed, which provides the basis for further study of possible roles of HSF2 in ulcerative colitis.