人CD63真核表达质粒的构建及在TCA8113细胞中的表达与定位  

Construction of eukaryotic expressing plasmid of human CD63 gene and its expression and location in TCA8113 cells

作　　者：刘文慧[1] 阚丽丽 侯美玲[1] 梁雪雪 赵微[1] 李新[3] 

机构地区：[1]辽宁医学院,辽宁锦州121200 [2]盘锦市辽河油田总医院,辽宁盘锦124000 [3]辽宁医学院附属第二医院,辽宁锦州121200

出　　处：《解放军医学院学报》2015年第8期836-839,共4页Academic Journal of Chinese PLA Medical School

基　　金：辽宁省博士启动基金(20141134);辽宁医学院校长基金-全民口腔研究生科研创新基金资助(QM2014003)~~

摘　　要：目的构建人CD63真核表达质粒,观察其在人舌鳞癌细胞TCA8113中的表达与定位。方法提取TCA8113细胞总RNA,RT-PCR方法扩增CD63全长基因序列,亚克隆至pc DNA3.1真核表达质粒中,构建pc DNA3.1-CD63表达质粒,转化E.coli DH5α,筛选阳性克隆,酶切及测序鉴定正确后,将重组质粒转染TCA8113细胞,Western blot检测蛋白表达,间接免疫荧光方法观察CD63蛋白在TCA8113细胞中的表达与定位。结果成功构建了pc DNA3.1-CD63真核表达质粒,Western bolt结果检测到外源CD63高表达,间接免疫荧光结果表明CD63融合蛋白与内源CD63蛋白定位相似,均表达于细胞膜。结论外源CD63融合蛋白能在TCA8113中高表达并定位于细胞膜。Objective To construct an eukaryotic expressing plasmid of human CD63 gene and observe its expression and location in TCA8113 tongue squamous cell carcinoma cell line. Methods Total RNA was extracted from TCA8113 cells. The CD63 gene was amplified by PCR method and subcloned to pc DNA3.1（＋） plasmid. The recombinant plasmids were transformed into E.coli DH5α and screened by restriction enzyme digestion and DNA sequencing. Then, pc DNA3.1-CD63 plasmids were transfected into TCA8113 cells. Western blot was used to detect the expression of protein, the subcellular location between endogenous CD63 and pc DNA3.1-CD63 in TCA8113 cells was detected and compared by indirect immunofluorescence staining. Results The recombinant eukaryotic expressing plasmid, pc DNA3.1-CD63, was successfully constructed. The expression of recombinant plasmid in TCA8113 cells was proved by Western blot. And the indirect immunofluorescence staining showed that the location of pc DNA3.1-CD63 protein was similar with endogenous CD63 protein, and they both located in the outer membrane of TCA8113 cells. Conclusion The fusion protein pc DNA3.1-CD63 can be successfully expressed in TCA8113 cells and located in the outer membrane.

关 键 词：CD63 真核表达 免疫荧光 舌癌 

分 类 号：R739.8[医药卫生—肿瘤]