KIAA1199基因重组慢病毒载体的构建及在人胃肿瘤细胞MKN28中的表达  

Construction of KIAA1199 recombinant lentivirus vector and its expression in human gastric carcinoma cell line MKN28

作　　者：蔡懿婷[1] 许慈[1] 董晴晴[1] 王碧君[1] 戴强[1] 朱黎明[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院(北部)消化科,上海201999

出　　处：《解剖学研究》2016年第5期375-379,共5页Anatomy Research

基　　金：上海交通大学医学院基金项目(14XJ10066);上海市宝山区科委科研基金项目(13-E-2)

摘　　要：目的构建KIAA1199基因过表达的重组慢病毒载体,感染人胃癌细胞MKN28并使KIAA1199基因有效表达。方法将慢病毒载体GV367和KIAA1199基因序列用Age I和Nhe I双酶切、连接、筛选及测序获得重组慢病毒质粒GV367-KIAA1199。将测序正确的GV367-KIAA1199重组载体和辅助质粒Help1.0和Help2.0用Lipofectamine2000共转染293T细胞,得到重组慢病毒载体LV-KIAA1199。予荧光稀释法和药物筛选法测定重组慢病毒的感染滴度。以携带空白基因片段的慢病毒载体LV-NC为病毒对照组。将LV-KIAA1199和LV-NC以相同的病毒感染复数(MOI)分别感染人胃癌细胞株MKN28;荧光显微镜观察EGFP在细胞中的表达;实时定量PCR检测KIAA1199的m RNA在细胞中的表达水平。结果重组慢病毒载体LV-KIAA1199构建成功,LV-KIAA1199感染胃癌细胞后,荧光显微镜下可见细胞中呈现绿色荧光;KIAA1199的m RNA的表达均显著高于对照病毒组。结论重组慢病毒载体LV-KIAA1199可携带KIAA1199基因在人胃癌细胞株MKN28中进行有效过表达。Objective To construct the recombinant lentivirus vector containing KIAA 1199 gene over expression. Human gastric carcinoma cell line MKN28 was infected by this recombinant adenovirus vector and the KIAA 1199 gene was expressed effec- tively in vitro. Methods The lentivirus vector plasmid GV367 and the KIAA1199 gene sequence were both digested by restriction enzymes with AgeIand NheI. The recombinant lentivirus plasmid GV367-KIAA1199 was generated by connection, selection and se- quencing. Thereafter, GV367-KIAA1199 was cotransfected with two assistant plasmids pHelp 1.0 and pHelp 2.0 into 293T cells to establish the recombinant lentivirusLV-KIAA 1199. The infective titer of recombinant lentivirus was tested by the methods of fluores- cence dilution and puromycin screen. Lentivirus LV-NC contains empty gene sequence was as lentivirus control group. The recombi- nant lentivirus LV-KIAA1199 and LV-NC were infected into human gastric cells MKN28 with the same Multiple of infection （MOI） re- spectively. Then the expression of EFGP was observed by fluorescence microscope. The mRNA expression of KIAA 1199 was testified by real time PCR in cells. Results The recombinant lentivirus LV-KIAA1199 was established successfully. The green fluorescence of EGFP was observed in MKN28 cells after LV-KIAA1199 infected the cells 24 h later. The gene expression of KIAA1199 was much more higher inLV-KIAA1199 treated group in MKN28 cells than in LV-NC group. Conclusion The target gene KIAAl199 can be contained by recombinant lentivirus vector and overexpressed effectively in human gastric carcinoma cell line in vitro.

关 键 词：KIAA1199 慢病毒载体 胃肿瘤 构建 表达 

分 类 号：R735.2[医药卫生—肿瘤]