【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pUASHA-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-HAh SNRPN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。更多还原

【Abstract】 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 pUASHA-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 U2 A 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, HAh SNRPN 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.更多还原