Nesprin2在小鼠睾丸各级生精细胞中的表达及其原核表达与纯化

The Expression of Nesprin2 in Different Stages of Germ Cells in Mouse Testis and Its Prokaryotic Expression and Purification

目的:研究Nesprin2在小鼠睾丸各级生精细胞中的表达变化,并对Nesprin2进行原核表达和纯化,为进一步研究该蛋白在精子发生中的作用奠定基础。方法:制备小鼠睾丸细胞涂片,用Nesprin2特异性抗体进行细胞免疫荧光染色,观察Nesprin2在各级生精细胞中的表达;应用RT-PCR技术扩增Nesprin2 C端包含KASH domain的编码85个氨基酸的目的片段,将PCR产物插入到PUCm-T载体中测序,将测序验证后的目的片段亚克隆至原核表达载体PGEX-4T-1中,转化大肠杆菌BL21,IPTG诱导表达。对GST-Nesprin2 C85融合蛋白进行纯化,Western blot进行验证。结果:免疫荧光检测结果发现,Nesprin2在小鼠睾丸各级生精细胞中均有表达,主要分布在核膜及其周围,在减数分裂过程中Nesprin2可能参与核膜重塑。构建了PGEX-4T-1-Nesprin2 C85重组质粒,经IPTG诱导后成功表达了GST-Nesprin2 C85融合蛋白。对GST-Nesprin2 C85融合蛋白进行纯化后,Western blot进行检测,结果发现,原核诱导表达的融合蛋白为GST-Nesprin2 C85融合蛋白。结论:Nesprin2在小鼠各级生精细胞中均有表达,在减数分裂过程中可能参与核膜的重塑。

Objective： To investigate the expression of Nesprin2 in different stages of mouse germ cells, and to obtain Nesprin2 fusion protein by prokaryotic expression and purification to further study its role in spermatogenesis. Methods： Mouse germ cell slides were prepared. Nesprin2 was detected in different stages of mouse germ cells with specific anti-Nesprin2 antibody by immunofluorescence staining. The target fragment in Nesprin2 C-end encoding 85 amino acids, which included a KASH domain, was amplified by RT-PCR. The PCR products were cloned into p UCm-T vector and sequenced, the verified target fragment was subcloned into p GEX-4T-1 vector, a prokaryotic expression vector with GST tag. The recombinant plasmid was then transformed into E.coli BL21.After induced by IPTG, the GST-Nesprin2-C85 fusion protein was purified by GST purification kit and identified by western blotting.Results： Immunofluorescence staining showed that Nesprin2 can be detected in all stages of mouse germ cells and was located in nuclear membrane and periphery. Nesprin2 may be involved in the remodeling of nuclear membrane in meiosis. The recombinant plasmid GST-Nesprin2-C85 was constructed and the fusion protein was expressed successfully after induced by IPTG. After purification, the protein was identified to be the GST-Nesprin2-C85 fusion protein as we expected by Western blotting. Conclusions： Nesprin2 is expressed in all stages of mouse germ cells, and it may participate in the remodeling of nuclear membrane. The constructed prokaryotic expression plasmid and the identified GST-Nesprin2-C85 fusion protein will lay a foundation for future research for finding its interaction protein by pull-down technique and interpreting its biological function in spermatogenesis.