HSV1-tk 报告基因真核表达载体的构建及其在人肺腺癌 AGZY 细胞中的表达  

Construction and expression of HSV1-tk eukaryotic vector in lung adenocarcinoma AGZY cell line

作　　者：栾厦[1] 付鹏[2] 金钟男[1] 田国梅[1] 姜廷军[1] 曹学良[1] 赵长久[1] 

机构地区：[1]哈尔滨医科大学附属第四医院核医学科,哈尔滨150001 [2]哈尔滨医科大学附属第一医院核医学科

出　　处：《实用肿瘤学杂志》2014年第2期97-101,共5页Practical Oncology Journal

基　　金：国家自然科学基金（81171362）;黑龙江省自然科学基金（D201060）

摘　　要：目的：构建含有HSV1-tk（ Herpes simplex virus type 1 thymidine kinase， HSV1-tk）基因的真核表达载体，并检测其在人肺腺癌AGZY细胞系中的表达。方法应用PCR反应从质粒pHSV106质粒中扩增HSV1-tk基因后与pMD18-T载体连接，构建重组质粒pHSV1-tk/18T。将测序正确的重组质粒插入plRES2-EGFP 载体内，通过LipofectamineTM 2000将表达载体转染人肺腺癌AGZY细胞。结果酶切鉴定结果表明扩增的HSV1-tk基因序列正确；用荧光显微镜观察HSV1-tk基因的转入和表达；RT-PCR和Western blot结果显示在AGZY细胞中HSV1-tk基因在转录水平和蛋白水平均可以正确表达。MTT结果显示转染后AGZY细胞与未转染细胞在细胞增殖能力方面无明显差别。结论成功构建HSV1-tk报告基因的真核表达载体，在人肺腺癌AGZY细胞中能有效表达。Objective The purpose of this study is to construct eukaryotic gene vector of herpes simplex virus type 1 thymidine kinase（HSV1-tk）and to observe the expression of HSV1-tk in lung adenocarcinoma AGZY cell line.Methods The full length HSV1-tk gene was amplified by PCR from plasmid pHSV 106 and was inserted into pMD18-T.The recombinant plasmid was recombined with eukaryotic vector plRES 2-EGFP u-sing gene recombinant technique .HSV1 -tk was transfected into adenocarcinoma AGZY cell line with Lipo-fectamineTM 2 000.Fluorescence microscopy was used to detect the transfection and expression of HSV 1-tk.RT-PCR was used to detect the mRNA levels of HSV 1-tk.The cell proliferation was measured by MTT assay .Re-sults A length of 1 130 bp gene sequence was obtained by PCR .The expressions of HSV 1-tk at mRNA and protein levels were displayed by RT -PCR and Western blot .MTT analysis showed that there were no significant changes cell survival on after transfection .Conclusion The eukaryotic expression vector of HSV 1 -tk report gene is successfully constructed and HSV 1-tk is effectively expressed in transfected AGZY cells .

关 键 词：HSV1-TK 真核表达载体 报告基因显像 肺腺癌AGZY细胞 

分 类 号：R734.2[医药卫生—肿瘤]