人CACNA2D3基因真核表达载体的构建及在食管鳞癌细胞中的表达  

Construction and Expression of CACNA2D3 in Esophageal Squamons Cell Carcinoma

作　　者：聂昌君 覃晓慧 钟青燕 王秋华 唐宁 蔡稔 曾定元 

机构地区：[1]柳州市出生缺陷预防与控制重点实验室,广西柳州545001 [2]柳州市妇幼保健院医学遗传科,广西柳州545001

出　　处：《现代生物医学进展》2017年第33期6442-6445,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81360365);广西省自然科学基金项目(2014GXNSFBA118205);广西壮族自治区卫生厅自筹经费课题(Z2013600)

摘　　要：目的:构建电压依赖型钙离子通道基因CACNA2D3(calcium channel,voltage-dependent,alpha 2 delta subunit 3)真核表达载体,为进一步研究其在食管鳞癌发生发展中的作用打下基础。方法:利用RT-PCR技术扩增CACNA2D3基因编码序列,并将其克隆到真核表达载体pc DNA3.1(+)载体中,经双酶切和测序鉴定后转染到人食管癌细胞系EC109中,通过QRT-PCR技术检测CACNA2D3基因的表达情况。结果:从正常组织c DNA中扩增出CACNA2D3基因片段,大小与预期一致;得到的pc DNA3.1-CACNA2D3重组质粒通过双酶切和电泳后符合预期片段,进一步测序鉴定显示插入片段序列与Gen Bank中CACNA2D3的序列一致;重组质粒转染食管鳞癌细胞EC109后CACNA2D3基因在转录水平表达明显增高。结论:成功构建了pc DNA3.1-CACNA2D3重组质粒,并完成了在食管癌细胞EC109中的外源表达,为进一步研究其生物学功能奠定基础。Objective： To construct the eukaryotic expression plasmid of human CACNA2D3 （calcium channel, voltage-depen- dent, alpha 2 delta subunit 3）in order to provide a basis for further study its role in the carcinogenesis and progression of esophageal squamous carcinoma. Methods： The coding sequence of CACNA2D3 was amplified by reverse transcription PCR and cloned into pcD- NA3.1 （＋） vector. The recombinant pasmid was identified by enzyme digestion and DNA sequencing analysis, then was transfeeted into esophageal carcinoma cell line （EC109）. The expression of CACNA2D3 in EC109 cells was detected by QRT-PCR. Results： Human CACNA2D3 gene was cloned from normal tissue cDNA. The amplified DNA fragment was in accordance with the expected length. The recombined clones were identified by agarose gel electrophoresis after restriction endonuclease digesting. The inserted sequence in re- combinant plasmid was the same as the sequence of CACNA2D3 cDNA published in GenBank. The EC109 cell line was transfection with pcDNA3.1-CACNA2D3. QRT-PCR showed that the expression of CACNA2D3 was increased after transfection. Conclusion： The human CACNA2D3 was successfully cloned into eukaryotic expression vector and could express in Esophageal squamous cancer cells. This work has laid the foundation for further study of the biological function of CACNA2D3.

关 键 词：电压依赖型钙离子通道基因 分子克隆 实时荧光定量PCR 

分 类 号：R33[医药卫生—人体生理学] Q78[医药卫生—基础医学]