坏死梭杆菌白细胞毒素部分融合基因的真核表达及生物学特性研究

Expression and characterization of the recombinant Leukotoxin fusion gene in in Pichia pastoris KM71H

为了解坏死梭杆菌白细胞毒素的致病机制,本研究将实验室前期构建的坏死梭杆菌白细胞毒素基因部分融合基因的真核表达质粒(p PIC9K-bsbse-gas-sh)转化毕赤酵母KM71H细胞,1%甲醇诱导其表达目的蛋白;SDS-PAGE结果显示,甲醇诱导3 d后,重组BSBSE-GAS-SH蛋白以分泌形式表达于培养物上清液中,分子量约为117.9 ku;western blot表明重组蛋白能够与抗BSBSE抗血清发生反应,具有良好的反应原性;细胞毒性试验表明重组蛋白对小鼠肝细胞和巨噬细胞均具有细胞毒性作用,并且具有剂量依赖性,其中重组蛋白对小鼠巨噬细胞的细胞毒性作用更强。本研究为揭示坏死梭杆菌白细胞毒素致病机制提供了相关的实验数据。

In order to study the pathogenesis of the leukotoxin of Fusobacterium necrophorum, the leukotoxin fusion gene fragment of bsbse-gas-sh were cloned into the eukaryotic expression vector pPIC9K to construct recombinant plasmid of pPIC9K- bsbse-gas-sh, which was transformed into Pichia pastoris KM71H. Then the recombinant leukotoxin protein （rBSBE-GAS-SH） was expression under methanol induction, and identified by SDS-PAGE and western blot. The results showed that the rBSBE-GAS-SH was secreted into the medium with molecular weight of about 117.9 ku. Moreover, cytotoxicity assay further indicated that the rBSBE-GAS-SH had cytotoxicity to mouse macrophage and liver cell in a dose-dependent manner, and the cytotoxicity of rBSBE-GAS-SH to mouse macrophage is higher than to mouse liver cells at the same concentration. These results lay the foundation to further study the pathogenesis of F.necrophorum leukotoxin.