人颗粒溶素基因真核表达载体的构建及在肝癌细胞中表达的实验研究  

Construction of eukaryotic expression vector containing human granulysin gene and its expression in hepatocellular carcinoma cell line

作　　者：缪家文[1] 李芳秋[1] 杨爱龙[1] 张春华[1] 

机构地区：[1]南京大学医学院临床学院(南京军区南京总医院)解放军医学检验中心分子生物学实验室,江苏南京210002

出　　处：《医学研究生学报》2005年第12期1073-1077,共5页Journal of Medical Postgraduates

基　　金：南京市科技发展计划自然科学基金资助项目(批准号:200401070-5)

摘　　要：目的:构建人颗粒溶素(GNLY)全长编码序列的真核表达载体,转染肿瘤细胞,观察GNLY在细胞内的表达及其对细胞增殖的抑制作用。方法:用RT-PCR技术获取人GNLY全长编码序列cDNA,克隆至pGEM-T载体进行测序,再将克隆片段插入质粒pcDNA3.1(+)中,构建真核表达载体pcDNA3.1(+)/GNLY,用阳离子聚合物转染试剂转染肝癌细胞系SMMC-7721,检测目的基因mRNA的表达,W ST-8法检测细胞增殖活性。结果:获得人GNLY全长编码序列cDNA,并成功构建真核表达载体pcDNA3.1(+)/GNLY;转染SMMC-7721细胞后,检测出目的基因mRNA的表达;重组质粒转染组与空质粒对照组细胞增殖活性有非常显著性差异(P<0.01和P<0.001),并且随着转染基因浓度的增加,细胞增殖活性逐渐降低,2.0μg/m l pcDNA3.1(+)/GNLY转染组细胞增殖活性下降至0.329。结论:pcDNA3.1(+)/GNLY真核表达载体构建成功,GNLY基因转染对SMMC-7721细胞能产生细胞毒性效应,对肝癌细胞具有一定的杀伤作用。Objective ：To clone human granulysin （GNLY） full-length eDNA, construct its eukaryotic expression vector and observe the expression and the effect of GNLY protein on the hepatocellular carcinoma cell line SMMC-7721. Methods：Full-length eDNA of GNLY was obtained by RT-PCR from human peripheral blood mononuclear cells, it was then inserted into pGEM-T Easy Vector and subcloned to pcDNA3.1 （＋） vector to construct recombinant eukaryotic expression vector pcDNA3.1 （＋）/GNLY. The recombinant plasmid was transfected into SMMC-7721 cells. Expression of GNLY gene in the transfectedceils was detected by RT-PCR and the viability of ceils was measured by WST-8 assay, Results：GNLY full-length cDNA was cloned and inserted into pcDNA3.1 （＋） vector. Expression of GNLY mRNA in the transfected SMMC-7721 cell s was confirmed with RT-PCR. In WST-8 assay, cell viability in the test group was significantly lower than those in the control group and decreased gradually with the increase of GNLY transfected concentration. Cell viability reached 0. 329 in 2.0 μg/ml pcDNA3.1 （＋）/GNLY transfected group. Conclusion：GNLY cDNA can be expressed in transfected SMMC-7721 cells and the expressed GNLY can significantly suppress the proliferation of SMMC-7721 cells.

关 键 词：颗粒溶素 真核表达 细胞抑制作用 WST-8 

分 类 号：Q782[生物学—分子生物学]