Thoc1真核表达载体构建及其与ClC-3蛋白在人宫颈癌细胞HeLa中的共定位观察  

Construction of Thoc1 eukaryotic expression vector,and its co-localization with ClC-3 in human cervical cancer HeLa cells

作　　者：刘学强 郑兆广 王汝上 钟杰 何桂芳 徐彬 LIU Xueqiang;ZHENG Zhaoguang;WANG Rushang;ZHONG Jie;XU Bin(School of Biosciences and Biopharmacentics, Guangdong Pharmaceutical University, Guangzhou 510006, China)

机构地区：[1]广东药科大学生命科学与生物制药学院,广州510006 [2]广州康臣药业有限公司肾病药物研究中心

出　　处：《山东医药》2018年第16期26-29,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81101666)

摘　　要：目的克隆Thoc1基因并构建pThoc1-DsRed2-N1真核表达载体,观察Thoc1和氯离子通道3(ClC-3)在人宫颈癌细胞HeLa中的共定位情况,分析ClC-3是否参与细胞转录过程。方法采用RT-PCR法扩增获得Thoc1基因的编码序列,将其克隆到p DsRed2-N1载体中,构建pThoc1-DsRed2-N1真核表达载体,经PCR、SacⅠ和Bam HⅠ双酶切、基因测序进行鉴定。将对数生长期HeLa细胞随机分为转染组、空转染组及空白对照组,分别采用Lipofectamine 2000转染pThoc1-DsRed2-N1质粒、p DsRed2-N1质粒及脂质体,转染48 h倒置荧光显微镜下观察红色荧光蛋白表达情况。取转染组细胞,采用免疫荧光技术观察Thoc1和ClC-3的共定位情况。结果 PCR鉴定结果显示在约2.1 kb处有一亮带;双酶切得到约4.7 kb和2.1 kb的两个片段,符合预期大小;基因测序结果显示与Genebank中Thoc1基因序列完全一致;证实成功构建pThoc1-DsRed2-N1真核表达载体。空转染组可观察到较强的红色荧光,空白对照组没有观察到红色荧光,转染组红色荧光强度介于空转染组与空白对照组之间;证实HeLa细胞中存在Thoc1表达。ClC-3分布于细胞核和细胞质中,主要是细胞核中;Thoc1只分布于细胞核中;两者叠加后在细胞核呈黄色荧光,证实ClC-3和Thoc1在HeLa细胞核上存在共同定位。结论成功构建了pThoc1-DsRed2-N1真核表达载体;Thoc1与ClC-3共定位于HeLa细胞核,提示ClC-3可能参与细胞转录过程。Objective To clone human Thoc1 gene and construct the eukaryotic expression vector pThoc1-DsRed2-N1,to observe the co-localization of Thoc1 with voltage-gated chloride channel 3（ ClC-3） in human cervical cancer HeLa cells,and to analyze whether ClC-3 is involved in the cell transcription. Methods Thoc1 coding sequence was amplified by RT-PCR and cloned into the p DsRed2-N1 vector to construct the eukaryotic expression vector pThoc1-DsRed2-N1,which was then identified by PCR,enzyme digestion（ SacⅠand Bam HⅠ）,and gene sequencing. HeLa cells in the logarithmic phase were divided into 3 groups： the transfection group,empty transfection group,and control group,which were transfected with pThoc1-DsRed2-N1 plasmid,p DsRed2-N1 plasmid,and lipidosome by Lipofectamine 2000,respectively.At 48 h,the gene expression was observed under inverted fluorescence microscope. The co-localization of Thoc1 with ClC-3 was detected by immunofluorescence in the the transfection group. Results PCR results showed that there was a 2. 1 kb target band,and meanwhile,4. 7 kb and 2. 1 kb bands were obtained by enzyme digestion. Sequencing proved that the sequence and splicing order of Thoc1 gene was matched with Genebank,suggesting that the fluorescent eukaryotic expression vector pThoc1-DsRed2-N1 was constructed successfully. Overt red fluorescence was observed in the empty transfection group. The red fluorescence intensity of the transfection group was between those of the empty transfection group and the blank control group,suggesting that Thoc1 was expressed in HeLa cells. ClC-3 was localized both in the nucleus and cytoplasm,mainly in nucleus. At the same time,Thoc1 was localized in the nucleus only,and yellow fluorescence was observed after we merged ClC-3 with Thoc1,indicating that ClC-3 and Thoc1 were co-located in the nucleus. Conclusion The eukaryotic expression vector pThoc1-DsRed2-N1 is successfully constructed,Thoc1 co-localizes with ClC-3 in the nucleus,suggesting that ClC-3 may be involved in the cell transcription p

关 键 词：Thoc1基因 真核表达载体 氯离子通道3 共定位 人宫颈癌细胞HeLa 

分 类 号：R34[医药卫生—基础医学]