小鼠microRNA-21慢病毒抑制载体的构建及鉴定

目的构建microRNA-21(miR-21)慢病毒抑制载体,为研究miR-21在小鼠体内的功能及作用机制打下基础。方法利用miR-21前体并将其克隆入LV3pGLV-H1-GFP质粒中,经酶切及测序鉴定,利用脂质体将鉴定的阳性重组LV3pGLV-H1-GFP-miR-21抑制载体、PG-P1-VSVG和pCMV-dR3个质粒转染到HEK-293T细胞,将所得病毒感染293T细胞,检测病毒滴度,并将制备的病毒颗粒感染乳鼠心肌细胞和尾静脉注射小鼠,检测miR-21在小鼠体内的表达。结果酶切及测序结果证明成功构建了LV3pGLV-H1-GFP-miR-21重组质粒,并成功包装成慢病毒,病毒滴度为2.4×109TU/ml,重组病毒成功感染心肌细胞,同时在Balb/c小鼠心脏有表达。结论成功构建miR-21慢病毒抑制载体,获得高效表达miR-21的慢病毒颗粒,为miR-21的进一步研究奠定了基础。

Objective Construct microRNA-21 (miR-21) inhibition of lentivirus for studying its function and mechanism of miR-21 in mice. Methods The precursor of miR-21 was cloned into the LV3 pGLV-H1-GFP. The LV3 pGLV-H1-GFP-miR-21 recombinant plasmids, PG-P1-VSVG and pCMV-dR were transfected HEK-293 T cells.Virus titer was detected after infection of 293 T cells.Finally we evaluated miR-21 expression after that the virus infected mice cardiomyocytes and intravenously injected mice. Results The results showed that LV3 pGLV-H1-GFP-miR-21 recombinant plasmid was successfully constructed.The lentivirus titer was 2.4×109 TU/ml.The recombinant virus successfully infected myocardial cells and Balb/c mice heart. Conclusion We successfully constructed miR-21 lentivirus and obtained expression of miR-21 lentivirus particles. That laid foundations for the function of miR-21.