PROKARYOTIC EXPRESSION, PURIFICATION AND IDENTIFICATION OF TWO HSP70s IN WUCHANG BREAM MEGALOBRAMA AMBLYCEPHALA
团头鲂(Megalobrama amblycephala)两种热休克蛋白70(HSP70s)的原核表达、纯化及鉴定

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.