人SNRPN基因在果蝇S2细胞中的功能研究  

Function of human SNRPN gene in Drosophila S2 cells

作　　者：于骏 武云浩 何辉 李志然 付杨博 陈霞[1,2] YU Jun;WU Yunhao;HE Hui;LI Zhiran;FU Yangbo;CHEN Xia(School of Medicine,Nantong University,Jiangsu 226001;Department of Obstetrics and Gynecology,the Second Affiliated Hospital of Nantong University)

机构地区：[1]南通大学医学院,江苏226001 [2]南通大学第二附属医院妇产科

出　　处：《交通医学》2021年第5期437-440,F0002,共5页Medical Journal of Communications

基　　金：南通市科技计划项目(JC2020024)。

摘　　要：目的:构建人SNRPN(hSNRPN)基因过表达质粒,观察在果蝇S2细胞中的作用,为男性不育症的功能保守性研究提供重要工具。方法:构建pUAS-HA-hSNRPN质粒,利用Ub-GAL4质粒在果蝇S2细胞中驱动pUAS-HA-hSNRPN的表达。采用Western blot和免疫荧光染色观察HA-hSNRPN融合蛋白的表达情况。通过免疫荧光染色和qRT-PCR检测hSNRPN基因对果蝇RNA剪接体亚基表达水平的影响。结果:本实验成功构建了pUAS-HA-hSNRPN过表达质粒,并利用Ub-GAL4在果蝇S2细胞中驱动HA-hSNRPN融合蛋白的表达。Western blot检测和免疫荧光染色结果均显示HA-hSNRPN融合蛋白在果蝇S2细胞中成功表达。免疫荧光染色结果显示,在果蝇S2细胞中异位表达hSNRPN基因可下调U2A蛋白。qRT-PCR结果提示,异位表达hSNRPN基因可下调果蝇RNA剪接体关键因子的表达水平。结论:本研究成功实现了HA-hSNRPN融合蛋白在果蝇S2细胞中的表达,hSNRPN可竞争性抑制果蝇RNA剪接体亚基的表达水平。Objective:To construct the over-expression plasmid of human SNRPN(hSNRPN)gene and observe its role in Drosophila S2 cells,so as to provide an important tool for the functional conservation research of male infertility.Methods:To construct the pUAS-HA-hSNRPN plasmid,and use Ub-Gal4 to drive the expression of pUAS-HA-hSNRPN in Drosophila S2 cells.The expression of HA-hSNRPN fusion protein is observed by Western blot and immunofluorescence staining.The effects of hSNRPN gene on the expression of Drosophila RNA splicing subunits are detected by immunofluorescence staining and qRT-PCR.Results:In this study,we successfully constructed the over-expression plasmid of pUAS-HA-hSNRPN and used Ub-gal4 to drive the expression of HA-hSNRPN fusion protein in Drosophila S2 cells.Western blot and immunofluorescence staining results showed that HA-hSNRPN fusion protein was successfully expressed in Drosophila S2 cells.Immunofluorescence staining showed that ectopic expression of hSNRPN gene could down-regulate the expression of U2A protein in Drosophila S2 cells.qRT-PCR results showed that ectopic expression of hSNRPN gene could also down-regulate the expression of key factors of RNA splicing factors in Drosophila.Conclusion:In this study,HA-hSNRPN fusion was successfully expressed in Drosophila S2 cells,and it was found that hSNRPN could competitively inhibit the expression of RNA splicing subunits in Drosophila.

关 键 词：SNRPN 果蝇S2细胞 U2A 剪接体 UAS-Gal4 

分 类 号：Q344.13[生物学—遗传学]