团头鲂（Megalobrama amblycephala）两种热休克蛋白70（HSP70s）的原核表达、纯化及鉴定

摘要

In this research, The fragments of Constitutive HSC70 and inducible HSP70 ORFs of Wuchang bream Megalobrama amblycephala were amplified by PCR, and then cloned into prokaryotic expression vector pET-22b（＋）. The two recombinant plasmids, confirmed by double-endonuclease digestion and DNA sequencing, were transformed into competent E. coli BL21 （DE3）. The two recombinant strains were induced by 1mmol/L IPTG at different temperatures and times, establishing optimal conditions for inducible expression. The expression products were purified by Ni-NTA His Bind Resins affinity chromatography and DEAE-Sepharose FF anion-exchange chromatography, and detected by SDS-PAGE and Western-blotting. The results show that two recombinant expression plasmids of pET-22b（＋）/Ma-HSC70 and pET-22b（＋）/Ma-HSP70, each expressing a 72kDa protein that could be recognized by rabbit source anti-HSP70 polyclonal antibody, were successfully constructed, and the purity of two purified fusion proteins was more than 95%. A higher temperature （37℃） was conducive to the rapid expression of fusion protein, which was easy to form inclusion body; a lower temperature （25℃） was in favor of the soluble expression of fusion protein, but also reduced the expression rate of fusion protein. All things considered, the optimal conditions for expressions of two fusion proteins of Ma-HSC70 and Ma-HSP70 are induced for 7h. at 25℃ and 30℃, respectively. In this study, two higher purified fusion protein of Ma-HSC70 and Ma-HSP70 have been obtained by prokaryotic expression of two HSP70s of Wuchang bream, thus laying foundation for further research on molecular structure and function of two proteins and associated antibodies.%采用PCR方法扩增团头鲂组成型HSC70和诱导型HSP70基因完整的编码区片段,并分别克隆到原核表达载体pET-22b（＋）中,然后转化大肠杆菌BL21（DE3）,用1mmol/L IPTG在不同温度及时间下进行诱导表达。采用Ni-NTA His Bind Resins亲和层析和DEAE-Sepharose FF阴离子交换柱层析对目的蛋白进行纯化,并进行SDS-PAGE和Western-blotting分析。结果表明,成功构建了团头鲂两种重组表达质粒pET-22b（＋）/Ma-HSC70和pET-22b（＋）/Ma-HSP70,表达融合蛋白的相对分子量均约为72kDa,并能与兔抗人HSP70多抗进行特异性结合,这两种HSP70s融合蛋白经纯化后的纯度均达到95%以上。本实验选择融合蛋白Ma-HSC70在25℃和Ma-HSP70在30℃下分别诱导7h作为可溶性表达的最佳条件。