鮰爱德华菌hcp基因的克隆及重组表达

通过克隆获得鮰爱德华菌Edwardsiella ictaluri LH51溶血素共调节蛋白(Hcp)编码基因hcp,该基因全长为489 bp,编码163个氨基酸,hcp编码蛋白的理论相对分子质量为17 800,等电点为5.21。氨基酸序列分析结果表明,鮰爱德华菌hcp基因与迟钝爱德华菌Edwardsiella tarda毒力蛋白EvpC(Hcp同源物)、发光杆菌Photorhabdus luminescens hcp基因等具有高度同源性。将鮰爱德华菌Hcp氨基酸序列连接至pGS-21a表达载体中,成功构建了pGS-21 a-hcp原核表达质粒;再将表达质粒转化至BL21(DE3)菌株后,经IPTG诱导、镍柱层析纯化获得大量携带GST和组氨酸双标签的融合蛋白。经SDS-PAGE和Western blot分析,确认获得了相对分子质量约为45 000的融合蛋白,并成功制备了具有较高效价的多克隆抗体。

Cloning and recombinant expression of pathogenic bacterium Edwardsiella ictaluri hcp gene

The hcp gene encoding hemolysin coregulated protein was sequenced from pathogenic bacterium Ed- wardsiella ictaluri LH51 by cloning. It was found that the hcp gene contained the opening reading frame of 489 bp in length, encoding 163 amino acids with molecular weight of 17 800, and theoretical isoelectric point of 5.21. Multiple sequence alignment indicated that the E. ictaluri hcp shared high homology with those from pathogenic bac- teria Edwardsiella tarda and Photorhabdus luminescens. A recombinant plasmid pGS-21a-hcp containing the cod- ing sequence of hcp gene was constructed using pGS-21a as a fused expression vector. The SDS-PAGE and West- ern blot revealed that the recombined protein was expressed successfully in Escherichia coli BL21 （ ED3 ） induced by IPTG. Then a recombinant fusion protein about 45 000 was purified by Ni2＋-NTA His-bind column chromatogra- phy, and the polyclonal antibody was prepared.