MiR-218慢病毒表达载体的构建及鉴定

摘要:构建人mir-218-2, pre-miRNA慢病毒表达载体，为研究miR-218在人体的功能及作用机制打下基础。以人hsa-mir-218-2前体序列，设计部分互补的正反向引物，进行引物退火，形成引物二聚体，PCR扩增引物二聚体，酶切后插入到线性化pGCSIL-GFP慢病毒表达载体中,对重组质粒进行双酶切鉴定，并进行慢病毒的包装与滴度检测。用构建好的慢病毒表达载体感染人胃癌细胞MKN-28，qPCR检测细胞内miR-218表达。结果显示，重组质粒经双酶切分析及转化菌液测序，插入序列正确，慢病毒表达载体感染人胃癌细胞后qPCR检测显示能显著增高miR-218的表达。说明本实验成功构建了hsa-mir-218-2慢病毒表达载体，感染人胃癌细胞后能有效提高miR-218的表达。为进一步研究miR-218在人体的功能及作用机制建立了实验基础。

Construction and identification of lentiviral expression vector of MiR-218

Abstract: To construct a lentiviral vector expressing Hsa-pre-mir-218-2 and make a foundation on studying of the functions and mechanisms of miR-218. Partially complementary Forward and reverse primers were designed based on the hsa-pre-mir-218-2 sequence. The primer dimers were generated by primer annealing method. These primer dimers were amplified by PCR and were inserted into enzyme-digested and linearized pGCSIL-GFP lentiviral vectors. The recombinant plasmid pGCSIL-GFP-mir-218-2 was identified by double enzyme digestion and then was packed with lentiviral packaging systems, and viral titer was determined. Human gastric cancer cells MKN-28 were infected with the constructed lentiviral vectors. The expression of miR-218 in MKN-28 cells was determined by qPCR. The double enzyme digestion and DNA sequencing analyses of the recombinant plasmid revealed that insertion element was correctly cloned into the vector. The qPCR demonstrated that the expression level of miR-218 in the lentivirus infected MKN-28 cells was increased significantly. It can effectively infect gastric cancer cells. The lentiviral vector with hsa-pre-mir-218-2 was constructed successfully and laid an experimental basis on the studies of the functions and mechanisms of miR-218.