重叠延伸PCR法构建人肿瘤坏死因子前体水解酶系列突变重组体及稳定转染HeLa细胞株的建立  

Construction of a Series of Recombinants of Mutated Human Tumor Necrosis Factor-α Converting Enzyme by the OE-PCR and Establishment of Stable Transfectant HeLa Cell Lines

作　　者：闫媛[1] 章杰[1] 杨渝珍[1] 过健俐[1] 李禹琼[1] 

机构地区：[1]华中科技大学同济医学院基础医学院生物化学与分子生物学系,武汉430030

出　　处：《华中科技大学学报（医学版）》2008年第1期1-4,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No30471963)

摘　　要：目的构建人肿瘤坏死因子前体水解酶(TACE)系列真核表达载体,转染HeLa细胞,建立稳定转染的HeLa细胞系。方法以THP1细胞cDNA为模板,PCR扩增人TACE基因的全长cDNA编码区序列,将其插入pMD18T载体中,构建出pMD18T-FL-TACE载体。在此基础上,应用重叠延伸PCR扩增出缺失解整合素区的TACE重组cDNA和缺失酶区的TACE重组cDNA,利用DNA重组技术将其分别定向插入真核表达载体pIRES2.0-EGFP中,同时TACE全长也构建入pIRES20.-EGFP。这3种真核表达载体经酶切和测序鉴定后,用脂质体法转染HeLa细胞,经过G418筛选,获得稳定转染的HeLa细胞株,采用RT-PCR、Westernblot及荧光法检测表达。结果成功构建了pIRES2.0-EG-FP/FL-TACE、pIRES20.-EGFP/disΔ-TACE、pIRES20.-EGFP/metΔ-TACE真核表达载体,并建立了稳定转染的HeLa细胞株,成功地表达了目的基因。结论真核表达载体的构建和稳定转染HeLa细胞株的建立为进一步研究TACE功能奠定了必要的实验基础。Objective To construct a series of eukaryotic expression vectors of human tumor necrosis factor-α converting enzyme（TACE）, and then to transfect HeLa cells with these vectors for establishment of stable HeLa cell lines. Methods The full-length TACE cDNA fragment was amplified by PCR from the human THP1 cell cDNA and subcloned into pMD18T vector to construct pMD18T-FL-TACE full-length vector. The cDNA fragment of disA-TACE and metA-TACE were amplified from plasmid pMD18T-FL-TACE full-length by using the overlap extension PCR and cloned into pIRES2.0-EGFP to construct the expression vector pIRES2.0-EGFP/FL-TACE, pIRES2.0 EGFP/disA-TACE and pIRES2.0-EGFP/metA-TACE. The fulllength of TACE was obtained by double digesting the plasmid pMD18T-FL-TACE full-length and then inserted into pIRES2.0-EGFP. These three eukaryotic expressing vectors were transfected into HeLa cells by LipofectamineTM 2000 after identification of digestion and sequencing. The stable transfected HeLa cell lines were then established by screening culture with G418, and the transcription and expression of TACE, disA-TACE and metA-TACE were identified by RT-PCR, Western blot and immu- nofluorescence. Results The eukaryotic expression vector pIRES2.0-EGFP/FL-TACE, pIRES2.0-EGFP/disA-TACE and pIRES2.0-EGFP/metA-TACE were constructed successfully, stable transfected HeLa cell lines were established, and the aim proteins were expressed successfully. Conclusion The construction of eukaryotic expression vector pIRES2.0-EGFP/FL-TA-CE, pIRES2.0-EGFP/disA-TACE, pIRES2.0-EGFP/met△-TACE and the establishment of stable transfected HeLa cell lines provided a solid experimental foundation for further studies on the function of the disintegrin domain of TACE.

关 键 词：人肿瘤坏死因子前体水解酶 解整合素 真核表达载体 重叠延伸PCR 转染 

分 类 号：Q7[生物学—分子生物学]