小鼠Tox3基因重组慢病毒载体的构建及其在卵巢颗粒细胞中的表达

Construction of the Recombinant Lentiviral Vector Containing Mouse Tox3 Gene and Its Expression in Primary Granulosa Cells

目的:构建重组慢病毒载体pCDH-Tox3-Flag,并检测其转染小鼠原代卵泡颗粒细胞后的表达情况。方法:利用DNA重组技术将小鼠Tox3基因克隆入慢病毒表达载体pCDH-MCS-T2A-copGFP-MSCV中,通过酶切、测序验证后,将重组慢病毒载体pCDH-Tox3-Flag、包装质粒psPAX2和包膜质粒pMD2.G共转染HEK293FT细胞,包装重组慢病毒LV-Tox3-Flag,并感染小鼠原代颗粒细胞,用反转录聚合酶链反应(RT-PCR)检测Tox3 mRNA的转录水平,蛋白质印迹(Western blot)法检测TOX3-Flag蛋白的表达情况。结果:经酶切及测序结果证实,构建了重组慢病毒载体LV-Tox3;RT-PCR及Western blot结果显示慢病毒感染小鼠原代颗粒细胞后,Tox3基因能在细胞内正确转录、翻译并稳定表达TOX3蛋白。结论:成功建立Tox3基因慢病毒表达载体LV-Tox3-Flag,包装的慢病毒能够成功感染小鼠原代颗粒细胞,并使Tox3基因得到稳定表达,为后续研究Tox3基因在生殖相关疾病的生理与病理作用奠定了基础。

Objective： To construct the recombinant lentiviral vector containing mouse Tox3 gene, and to check its expression in primary granulosa cells. Methods： The full length of Tox3 fragment was amplified by PCR, and subcloned into the lentiviral vector pCDH-MCS-T2A-copGFP-MSCV. The resultant lentivirus were confirmed by PCR, restriction enzyme digestion and DNA sequencing. The recombinant lentivirus （LV-Tox3- Flag） were produced from HEK293FF by a transient co-transfeetion with psPAX2 and pMD2.G. Primary granulosa cells were infected by LV-Tox3-Flag lentivirus, and the expression of TOX3 was confirmed by RT- PCR and Western blot. Results： The recombinant lentiviral vector carried the Tox3 gene was successfully constructed. RT-PCR and Western blot analysis revealed that the Tox3 gene can be correctly transcript and translated in the granulosa cells infected by LV-Tox3. Conclusions： The recombinant LV-Tox3-Flag was successfully constructed. It will be used to transfect target cells in our future study.