基于GatewayTM系统Dec1慢病毒表达载体的构建

摘要

Objective To construct lentiviral expression vector of human differentially expressed in chondrocytes (Decl) by Gateway technology and establish stable expressing target gene of human glioma U87 cell lines. Methods Total RNA was extracted from human glioma tissues and reversed transcript to obtain object cDNA. Primers containing specific enzyme sites were designed and used to amplify full-length of target gene Decl for recombination by reverse transcriptase-polymerase chain reaction (RT-PCR). The target fragment was cloned into entry vector (pENTRTM 3C) to generate an entry clone. Target gene in entry clone was transferred into destination vector (pLenti 6. 3/V5-DEST) to generate an expression clone pLenti6. 3-Decl by GatewayTM technology. The lentiviral expression vector pLenti6.3-Decl was packaged in 293T cells and transfected into U87 cells. U87 cell lines stable expressing Decl were obtained by Blasticidin screening and Western blot identification. Results Obtaining of full-length of Decl gene and constructing of lentiviral expression vector pLenti6.3-Decl were confirmed by PCR and sequencing. U87 cell lines with stable expressed Decl were obtained by Blasticidin screening after being transfected with the pLenti6. 3-Decl. Conclusion Lentiviral expression vector pLenti6.3-Decl is successfully constructed and U87 cell lines with stable expressed Decl are established, which lays a foundation for further exploring molecular mechanism of Decl.%目的 利用GatewayTM技术构建分化型胚胎软骨发育基因1(Dec1)的慢病毒表达载体并建立稳定表达该目的基因的人胶质瘤U87细胞株.方法 从人脑胶质瘤组织中提取总RNA,经反转录得到目的cDNA.设计含有特异性酶切位点的引物,通过逆转录酶-多聚酶链反应(RT-PCR),获得用于重组的Dec1目的基因全长.将目的片段克隆至入门载体(pENTRTM 3C)中产生入门克隆.利用GatewayTM技术,将入门克隆中的目的片段转接于目的载体(pLenti6.3/V5-DEST)中产生表达克隆pLenti6.3-Dec1.在293T细胞中包装慢病毒表达载体pLenti6.3-Dec1并转染U87细胞,通过灭瘟素筛选、Western blot鉴定,获得稳定表达Dec1的U87细胞株.结果 Dec1基因全长的获取与慢病毒表达载体pLenti6.3-Dec1的构建经PCR和测序得到证实.用pLenti6.3-Dec1转染U87细胞后,通过灭瘟素筛选,获得稳定表达Dec1的U87细胞株.结论 成功构建了pLenti6.3-Dec1慢病毒表达载体并建立稳定表达Dec1的人胶质瘤U87细胞株,为深入研究Dec1分子机制奠定了基础.