牙鲆(Paralichthys olivaceus)CD59基因的原核表达与功能探究

Prokaryotic Expression and Functional Analysis of CD59 of Japanese Flounder(Paralichthys olivaceus)

本研究分析了CD59基因在牙鲆(Paralichthys olivaceus)感染鳗弧菌(Vibrio anguillarum)前后的表达模式。结果显示,感染前,CD59基因在检测的4种免疫器官中均不表达;感染后,该基因均有不同程度的表达,其中,肾脏的表达量最高。对牙鲆CD59基因进行原核表达,构建原核表达载体pET-32a-CD59,并将其转化入大肠杆菌(Escherichia coli)BL21,使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导,获得约为29 kDa的重组蛋白pET-32a-CD59。该蛋白以包涵体形式存在,通过His亲和层析柱纯化和超滤管浓缩目的蛋白后,经SDS-PAGE检测得到单一条带,纯化效果较好。Western blotting分析结果显示,重组蛋白p ET-32a-CD59可以与抗His单克隆抗体发生特异性反应,说明牙鲆CD59基因在大肠杆菌系统中成功表达。对重组蛋白进行透析复性,复性结束后,蛋白无析出或絮状沉淀,初步表明复性成功。选取嗜水气单胞菌(Aeromonas hydrophila)、鳗弧菌、金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌4种致病指示菌,测定复性重组蛋白p ET-32a-CD59的抑菌活性。研究表明,重组蛋白pET-32a-CD59对嗜水气单胞菌具有一定的抑菌活性。本研究旨在探究牙鲆免疫调节机制,并为提高牙鲆养殖效率提供一定的分子理论基础。

CD59 is a glycophosphatidylinositol （GPI）-linked protein, identified as a regulator of the terminal pathway of complement membrane system. This study reported the gene expression patterns of CD59 of Japanese flounder （Paralichthys olivaceus） before and after challenged with Vibrio anguillarum. The results implied that few expression of CD59 was detected in all tissues before challenged with V. anguillarum. After being challenged, the expression levels of CD59 were significantly higher than the control group on different levels. And the most abundant expression was identified in kidney. Meanwhile the CD59 gene was inserted into the pET-32a to construct prokaryotic expression plasmid. The recombinant plasmid expressed in Escherichia coli BL21 （DE3） cells through isopropyl-β-thiogalacto- pyranoside （IPTG） induction and approximate 29 kDa recombinant protein of pET-32a-CD59 was obtained. Through the analysis of ultra-sonication on recombinant expression strains, the recombinant pET-32a-CD59 was identified in precipitate. It implied that the recombinant pET-32a-CD59 existed as an inclusion body. Then the recombinant protein was purified and concentrated by Ni-chelating affinity chromatography and ultrafiltration. SDS-PAGE analysis revealed a single band implying successful purification. Western blotting analysis showed that the recombinant protein could be specifically combined with anti-His monoclonal antibody. So the expressed protein was confirmed as the target protein. The recombinant protein was refolded by dialysis because of its incapability in biological activity. There was no precipitate or separation after refolding, which suggested that the operation of refolding by dialysis on recombinant protein was successful. After purification and refolding, the antimicrobial activity of the recombinant CD59 was demonstrated in Japanese flounder. Four pathogens were chosen for the experiment including Aeromonas hydrophila, V. anguillarum, Staphylococcus aureus and E. coli. The results revealed that recombinant Japanese flounder CD59 displayed antimicrobial ability through the inhibition on the growth of A. hydrophila. This study explored the immune regulation mechanism in Japanese flounder and provided molecular theoretical basis for improving the efficiency of breeding in Japanese flounder.