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 .
出处
《实用肿瘤学杂志》
CAS
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）
关键词
HSV1-TK
真核表达载体
报告基因显像
肺腺癌AGZY细胞
Herpes simplex virus type 1 thymidine kinase
Eukaryotic expression vector
Reporter geneimaging
Lung adenocarcinoma AGZY cell
