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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 a recombinant lentiviral vector overexpressed KIAA1199 and infect human gastric cancer cell line MKN28 and express KIAA1199 gene efficiently. Methods The lentiviral vector GV367 and KIAA1199 were digested by Age I and Nhe I, ligated, screened and sequenced to obtain the recombinant lentiviral plasmid GV367-KIAA1199. The correct sequencing of GV367-KIAA1199 recombinant vector and helper plasmids Help1.0 and Help2.0 Lipofectamine2000 co-transfected 293T cells, to obtain a recombinant lentiviral vector LV-KIAA1199. The titer of recombinant lentivirus was determined by fluorescent dilution and drug screening. The lentiviral vector LV-NC carrying the blank gene fragment was used as a virus control group. The human gastric cancer cell line MKN28 was infected with LV-KIAA1199 and LV-NC at the same multiplicity of infection (MOI), respectively. The expression of EGFP in the cells was observed by fluorescence microscopy. The expression of m RNA in KIAA1199 cells was detected by real-time quantitative PCR. Results The recombinant lentiviral vector LV-KIAA1199 was successfully constructed. After LV-KIAA1199 was infected in gastric cancer cells, the cells showed green fluorescence under the fluorescence microscope; the expression of m RNA of KIAA1199 was significantly higher than that of the control group. Conclusion The recombinant lentiviral vector LV-KIAA1199 can efficiently transfect KIAA1199 gene in human gastric cancer cell line MKN28.