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目的:构建携带人ILT3基因重组慢病毒载体并检测其生物学功能。方法:从已构建好的含ILT3的质粒pB lue-scriptR-ILT3为模板,利用聚合酶链反应(PCR)方法扩增目的基因ILT3;将该基因与慢病毒载体表达质粒pLVX-AcGFP-N1连接,得到重组的pLVX-AcGFP-ILT3,通过酶切和测序分析比对验证ILT3基因后,将pLVX-AcGFP-ILT3质粒利用慢病毒转染系统转染人胚胎肾上皮细胞株Lenti-X 293T细胞获得携带ILT3慢病毒载体;转染人脐静脉血管内皮细胞,利用流式细胞仪检测目的基因ILT3的表达。结果:经PCR扩增获得1 844 bp的目的基因片段,构建的重组质粒经酶切及测序和分析比对鉴定正确;该质粒与包装质粒共转染293T细胞获取的慢病毒,纯化、浓缩后滴度达5×1012转导单位pfu/L;转染的HUVECs,流式细胞检测ILT3阳性率为98%,可判断目的基因ILT3在靶细胞HUVECs中表达。结论:成功构建转载质粒pLVX-AcGFP-ILT3及携带ILT3基因慢病毒载体。
Objective: To construct a recombinant lentivirus vector carrying human ILT3 gene and test its biological function. METHODS: ILT3 gene was amplified from the constructed plasmid pBue-scriptR-ILT3 containing ILT3 by polymerase chain reaction (PCR) and ligated with lentiviral vector plasmid pLVX-AcGFP-N1 , And the recombinant pLVX-AcGFP-ILT3 was obtained. The ILT3 gene was verified by restriction analysis and sequencing analysis, and then the pLVX-AcGFP-ILT3 plasmid was transfected into the human embryonic kidney cell line Lenti-X 293T cells by lentiviral transfection system Carrying ILT3 lentiviral vector; transfecting human umbilical vein endothelial cells, and detecting the expression of ILT3 gene by flow cytometry. Results: The gene fragment of 1 844 bp was obtained by PCR amplification. The constructed recombinant plasmid was identified by restriction enzyme analysis, sequencing and analysis. The recombinant plasmids were co-transfected with the packaging plasmid to obtain the lentivirus, purified and concentrated The titer was 5 × 1012 transduction unit pfu / L. The positive rate of ILT3 in transfected HUVECs was 98%. The expression of ILT3 in HUVECs could be determined. Conclusion: The recombinant plasmids pLVX-AcGFP-ILT3 and ILT3 gene lentiviral vectors were successfully constructed.