鸡β-防御素-3在毕赤酵母中的分泌表达及其生物活性测定

Secreted expression of chicken β-gallinacin-3 in Pichia pastoris and biological activities detection

采用RT-PCR方法从鸡外周血淋巴细胞中扩增鸡β-防御素-3成熟肽基因,将其克隆到酵母表达载体pPICZα-A的分泌信号肽基因下游。线性化后的重组质粒电击转化毕赤酵母宿主菌GS115,经抗生素Zeocin筛选和PCR鉴定后,得到重组酵母菌。阳性转化菌经甲醇诱导后,SDS-PAGE和western blot鉴定结果表明,在毕赤酵母中得到分泌性表达产物,分子量约为8.9ku,活性检测表明其具有显著的抑菌活性。

The gal-3 gene was amplified from total RNA of chicken peripheral blood lymphocytes by RT-PCR and subcloned into the Pichia pastoris expression vector pPlCZα-A. The recombinant plasmid was transformed into P.pastoris strain GS115 by electroporation. Recombinant strains were screened on YPDS plates containing Zeocin and identified by PCR. Exepression of gal-3 were induced by methanol. SDS-PAGE and western blot analysis showed that recombinant protein was expressed in soluble form with molecular weight approximately 8.9 ku. The expressed Gal-3 protein showed excellent antibacterial activity against E.coli and S.aureus in vitro antibacterial assays.