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目的:构建人NYD-SP28基因原核表达载体,表达其原核蛋白,制备其多克隆抗体,以探讨其在精子发生中的作用。方法:以原始质粒为模板,用PCR法扩增人的NYD-SP28基因开放阅读框全长,将其克隆到原核表达质粒pET28a(+)中,构建重组质粒pET28a-NYD-SP28。将该重组质粒转化大肠杆菌BL21,以IPTG诱导原核蛋白的表达,纯化蛋白以免疫小鼠,制备多克隆抗体,用ELISA法测定抗体的滴度。结果:成功构建了用于原核蛋白表达的重组质粒,并在BL21菌中表达NYD-SP28原核蛋白,用亲和层析Ni柱进行纯化得到NYD-SP28原核蛋白。用纯化的NYD-SP28蛋白免疫小鼠2个月后,得到相应的多克隆血清抗体,经ELISA检测,滴度达到1/10万。结论:成功构建人NYD-SP28原核表达载体,并获得了高纯度的NYD-SP28蛋白及鼠抗NYD-SP28多克隆抗体,为进一步研究NYD-SP28的功能奠定了基础。
OBJECTIVE: To construct prokaryotic expression vector of human NYD-SP28 gene and express its prokaryotic protein to prepare its polyclonal antibody to explore its role in spermatogenesis. Methods: The full-length open reading frame of human NYD-SP28 gene was amplified by PCR from the original plasmid and cloned into prokaryotic expression plasmid pET28a (+). The recombinant plasmid pET28a-NYD-SP28 was constructed. The recombinant plasmid was transformed into E. coli BL21, IPTG induced prokaryotic protein expression, purified protein to immunize mice to prepare polyclonal antibodies, antibody titer was measured by ELISA. Results: The recombinant plasmid for prokaryotic expression was successfully constructed. NYD-SP28 prokaryotic protein was expressed in BL21 and purified by affinity chromatography on Ni column to obtain NYD-SP28 prokaryotic protein. After two months of immunization of the mice with the purified NYD-SP28 protein, the corresponding polyclonal serum antibodies were obtained and the titer reached 1/100000 by ELISA. CONCLUSION: The NYD-SP28 prokaryotic expression vector was successfully constructed and NYD-SP28 protein and mouse anti-NYD-SP28 polyclonal antibody were obtained, which laid the foundation for further study on the function of NYD-SP28.