miR-196b海绵吸附慢病毒载体的构建及其在骨髓基质细胞系ST2中的表达

Construction of miR-196b sponge lentiviral vector and its expression in bone marrow stromal cell line ST2

目的构建miR-196b海绵吸附慢病毒载体,为研究miR-196b在骨髓基质细胞系中的功能奠定基础。方法根据miR-196b的成熟体序列设计与之互补的串联的六重复序列设计成引物,通过PCR反应将该六重复序列亚克隆至pUC19质粒中,再经酶切连接至pLVX-shRNA2慢病毒载体,利用293T细胞将构建成功的miR-196b海绵吸附慢病毒载体进行病毒包装和滴度测定,pLVX-shRNA2慢病毒作为对照组,miR-196b海绵吸附慢病毒作为实验组,将其感染ST2细胞,观察感染效率,并通过Western blotting检测miR-196b靶基因叉头框蛋白O1(FoxO1)的蛋白水平。结果酶切鉴定和测序结果正确,慢病毒包装所测滴度为1×108PFU/mL,将其感染靶细胞,其感染效率达80%。Western blotting结果显示,与对照组相比,其靶基因FoxO1蛋白表达水平有明显增加(P<0.05)。结论成功构建了miR-196b海绵吸附慢病毒载体,并能有效吸附内源性的miR-196b,从而发挥其抑制作用。

Objective To construct miR-196b sponge lentiviral vector,and laid the foundation for studying the function of miR-196b in bone marrow stromal cells.Methods Based on the miR-196b mature sequence,a sequence consisting of6tandem repeats of the complementary sequence of miR-196b was designed,and which was cloned into pUC19plasmid by using reverse PCR.Then the six-repeat sequence was cut and subcloned into pLVX-shRNA2lentiviral vector.The lentivirus was packaged using293T cells,and titer determination was done.The pLVX-shRNA2lentivirus was used as the control group,and the196b-sponge-pLVX lentivirus was the experimental group.Then ST2cells were infected with the viruses,and the infection efficiency was calculated.The protein level of forkhead box O1(FoxO1)was detected by Western blot assay.Results The identity of the sponge sequence was verified by sequencing.The titer of the sponge virus was1×108PFU/mL,and the infection efficiency reached80%.Compared with the control group,the expression level of FoxO1protein was significantly increased(P<0.05).Conclusion The miR-196b sponge lentiviral vector is successfully constructed,and which has the capability to inhibit endogenous miR-196b.