miR-186过表达慢病毒载体的构建及鉴定

doi:10.13481/j.1671-587x.20190504

Abstract

Abstract: Objective:To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and toexplore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods:The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoRⅠand AgeⅠrestriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results:The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×10⁸ TU·mL^-1 and 5×10⁸ TU·mL^-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12.640 0±0.788 4) was significantly increased by 15.07 times (t=14.72,P<0.01) compared with control group (0.838 7±0.145 6). Conclusion:The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus suceessfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.

Key words: miR-186, lentivirus, HEK293T cells, overexpression lentiviral vector, green fluorescence protein

CLC Number:

R346

LI Shengnan, WANG Mengxu, HU Weidong, CHEN Shaofeng, CHEN Xinglan, LI You. Construction and identification of miR-186 overexpression lentiviral vector[J].Journal of Jilin University(Medicine Edition), 2019, 45(05): 997-1002.

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Construction and identification of miR-186 overexpression lentiviral vector

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