miR-494慢病毒表达载体的构建

Construction of a lentiviral expression vector harboring miR494

目的:构建miR-494基因的慢病毒表达载体。方法:以人基因组DNA为模板,PCR扩增hsa-miR-494基因,用XhoⅠ、BamHⅠ双酶切携带EGFP的慢病毒载体pGIPZ,酶切产物电泳后回收约11kb的载体片段。使用DNA连接试剂盒中的Solution I将其与miR-494连接,连接产物转化,次日挑选单菌落,PCR筛选正向阳性克隆,随后将选定的含有阳性克隆的单菌落摇菌,提取质粒并行酶切鉴定及对插入的miR-494测序,所构建载体命名为pGIPZ-miR-494-eGFP,获pGIPZ-miR-494-eGFP后按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产。将慢病毒颗粒以最适滴度感染人体肺腺癌细胞株A549,通过Aldefluor流式分选出带有目的基因和空载病毒的细胞,用Real-Time PCR检测感染效率。结果:目的基因与慢病毒载体连接成功。共转染293T细胞包装病毒与浓缩后滴度达1.02×108TU/ml,并转染到肺腺癌细胞株A549上,Aldefluor流式分选出的细胞后通过Real-Time PCR检测结果显示miR-494慢病毒表达载体感染的A549细胞miR-494表达高于对照组达10倍以上。结论:成功构建携带人miR-494基因的慢病毒表达载体pGIPZ-miR-494-eGFP,为相关后续研究打下了良好基础。

Objective：To construct lentivirus expression vector miR -494 gene. Methods：Human genomic DNA template, PCR amplification of hsa - miR - 494 gene. To use Xho Ⅰ and BamHⅠ double digested carrying the EGas a FP lentiviral vector pGIPZ of recovered after electrophoresis of the digestion products of the vector fragment of approximately 11 kb. Using a DNA ligation kit Solution I connected the miR -494 connection, the ligation product was transformed the next day, the selection of single colonies, PCR screening positive clones, containing the positive clones was selected single colonies were shake bacteria the extracted plasmid parallel restriction enzyme digestion and sequencing of the insertion of miR - 494, named pGIPZ - miR - 494 - eGFP, the standard procedure was recommended by Invitrogen Corporation pGIPZ - miR - 494 - eGFP. Lentiviral vector particles the optimal titer infection with the human lung adenocarcinoma cell line A549, Sub - elected with a target gene and no - load virus cells through Aldefluor flow cytometry, using Real - Time PCR detected infection efficiency. Results： A successful connected with the target gene with the lentiviral vector. Co - transfected 293T cell packaging virus and concentrated to titers of up to 1.02×10^8TU/ ml, transfected into A549 through Aldefluor flow eytometry separated target gene and no - load virus cells. The Real - Time PCR results showed miR-494 lentiviral vector- infected A549 cells higher than control group 10 times more. Conehmion：Suceessfully build miRNA lentivirus vector, packaging high -titer lentiviral, laid the foundation for the subsequent infection of lung cancer cells, to explore its role in the occurrence and development of lung cancer.