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

LIU Xueqiang; ZHENG Zhaoguang; WANG Rushang; ZHONG Jie; HE Guifang; XU Bin

doi:10.3969/j.issn.1002-266X.2018.16.007

Abstract：Objective To clone human Thoc1 gene and construct the eukaryotic expression vector pThoc 1-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 pDsRed2-N1 vector to construct the eukaryotic expression vector pThoc 1-DsRed2-N1, which was then identified by PCR,enzyme digestion(SacⅠand BamHⅠ),and gene sequencing.HeLa cells in the loga-rithmic phase were divided into 3 groups:the transfection group,empty transfection group,and control group,which were transfected with pThoc1-DsRed2-N1 plasmid,pDsRed2-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 se-quence 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 cyto-plasm,mainly in nucleus.At the same time,Thoc1was 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 eu-karyotic 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 process.