利用枯草杆菌整合表达嗜铬粒蛋白抗真菌片段

为实现CGA1-76片段基因在枯草杆菌中的稳定表达,将CGA1-76片段基因的表达元件重组到枯草杆菌转座子质粒pHV1249微转座子mini-Tn10内,利用mini-Tn10将CGA1-76片段基因的表达元件整合到枯草杆菌DB1342染色体上,用氯霉素和红霉素筛选枯草杆菌转座工程菌DB1342(TnSVTQ).DB1342(TnSVTQ)在无选择压力条件下经10 d连续传代培养,转座子内氯霉素抗性丢失率为0%,说明转座子稳定整合在DB1342染色体上.SDS-PAGE和免疫印迹分析结果表明,在蔗糖诱导下,CGA1-76片段基因在DB1342(TnSVTQ)中获得表达,产物被分泌到细胞外.孔穴琼脂扩散法测定表达上清的抑制真菌活性,结果表明重组CGA1-76能抑制烟曲霉菌和黄曲霉菌的生长,抑菌圈直径分别为9 mm和8 mm.

Expression of N-domain of Chromogrnin A in B.Subtilis by Mini-Tn10 Transposition and Examination of Its Antifungal Activity

To ensure the stable expression of CGA1-76 in B.subtilis, the expression cassette of CGA1-76 was inserted into mini-Tn10 of pHV1249 and was transposed into the chromosome of B.subtilis DB1342 resulting in the engineered strain B.subtilis DB1342(TnSVTQ) which has only chloramphenicol resistance and lose erythromycin resistance. DB1342(TnSVTQ) was still stable after 10 generations of growth in LB medium without selection stress. CGA1-76 was expressed in DB1342(TnSVTQ) by sucrose induction and secreted into the medium as shown by SDS-PAGE and western blot. The antifungal activity of expressed product was examined, and the result showed that the expressed product can inhibit the growth of Aspergillus fumigatus and Aspergillus flavus with inhibition zones 9 mm and 8 mm, respectively.