NYD-SP15基因的原核表达及多克隆抗体的制备

Prokaryotic expression plasmid of NYD-SP15 and preparation of polyclonal antibody

目的:克隆NYD-SP15基因,构建融合表达载体,在大肠杆菌中表达,并制备抗体。方法:PCR技术扩增NYD-SP15全长开放阅读框,克隆入原核表达载体PET28a,转化大肠杆菌感受态细胞,异丙基硫代-β-D-半乳糖苷(IPTG)诱导后收集菌体蛋白,行SDS-PAGE电泳。将此融合蛋白Ni柱纯化后免疫BALB/C小鼠,Western blotting鉴定。结果:成功地构建了PET28a-NYD-SP15原核表达质粒,并获得了高效表达NYD-SP15的BL21菌株,表达的His标签融合蛋白,分子量为58kDa左右,经免疫小鼠获得了抗NYD-SP15抗体。经Western blotting法分析,抗体为NYD-SP15特异性抗体。结论:构建的NYD-SP15基因的原核表达载体,体外高效表达蛋白,免疫小鼠获得抗NYD-SP15抗体,将为眼部增殖性视网膜病变(PVR)的研究及治疗奠定坚实的基础。

AIM ： To construct the prokaryotic expression plasmid of NYD-SP15 which is related to proliferative lesion, induce protein expression in vitro and immunize mouse for antibody. METHODS： Open-reading frame of NYD-SP15 gene was amplified by PCR technique, then we inserted it into PET28a prokaryotic expression vector and transformed into expressing strain. Its protein expression was induced by isoproprlthiogalactoside （IPTG）, then purified by Ni stele. Eight- week-old BALB/C mice were immunized by the purified protein to obtain antibody. The protein and its antibody were checked by Western blotting technique. RESULTS： We had successfully constructed NYD-SP15＇ prokaryotic plasmid, and the BL21 strain that highly expressed NYD-SP15 protein. The molecular weight of this His tagged-fusion protein was about 58 kDa. Polyclonal antibody was gained from immune mouse and the specificity was determined by Western blotting. CONCLUSION ： NYD-SP15 prokaryotic expression vector, the highly expression protein in vitro and its polyclonal antibody may establish a substantial foundation for the research and treatment of the proliferative retinopathy.