恒河猴miR-125a-3p慢病毒载体的构建、鉴定以及功能研究

Construction,identification and function of recombinant lentivirus vector for rhesus miR-125a-3p

构建携带miR-125a-3p过表达与抑制表达shRNA的慢病毒,研究miR-125a-3p对轮状病毒感染乳鼠导致的腹泻的影响,为进一步研究miR-125a-3p对轮状病毒增殖过程中的影响及探究其机制提供前期研究基础。设计miR-125a-3p前体序列及其反向互补序列,与慢病毒骨架质p LVshRNA-EGFP(2A)Puro载体进行酶切连接,构建过表达与抑制表达miR-125a-3p的慢病毒表达载体。利用HEK293Ta细胞包装慢病毒颗粒,测定病毒滴度后,将获得的miR-125a-3p过表达慢病毒颗粒感染MA104细胞和灌胃感染乳鼠,检测miR-125a-3p的表达。同时对乳鼠进行轮状病毒攻毒试验,观察腹泻情况。研究成功构建了过表达及抑制miR-125a-3p表达的慢病毒载体,获得高效的过表达及抑制miR-125a-3p的慢病毒颗粒,成功感染MA104细胞,以及在乳鼠小肠组织有表达。同时发现miR-125a-3p过表达慢病毒感染乳鼠后对轮状病毒引起的腹泻有明显的抑制作用。

To construct miR-125a-3p overexpressiou and suppression lentivirus shRNA vectors for studying its effect to diarrhea of m!R-125a-3p in mice, which laid a foundation of study miR-125 a-3 p to provide preliminary preparation for the impact of rotavirus prolif- eration process and to explore the molecular mechanism, the miR-125a-3p precursor sequence and miR-125a-3p reverse complement sequence were designed, and pLVshRNA-EGFP（2A）Puro vector was used to construct recombination lentivirus plasmid. The recombi- nant plasmid can generate the miR-125a-3p overexpression and suppression lentivirus vectors. Packaged lentivirus particle contained miR-125a-3p overexpression and suppression lentivirus by HEK293Ta. Virus titer was detected after infection of HEK293Ta. Finally we evaluated miR-125a-3p expression after that the virus infected MA104 cell and injected mice, and diarrhea of mice that was infected by rotavirus. It is successfully to construct the miRNA-125-3p overexpression and suppression lentivirus vectors. The recombinant virus successfully infected MAIIM cells and mice small intestine, miR-125a-3p overexpression and suppression lentivirus vector were con- strueted successfully, which provide a stable cell transfection vector for further study of the impact and mechanism of miR-125a-3p to ro- tavirus and its host. We also found that the miR-125a-3p overexpression lentivirus vector can inhibitor the diarrhea,which was infected by rotavirus.