少孢节丛孢菌Aoz1基因的生物信息学分析及原核表达条件的优化

Bioinformatics analysis of Aoz1 gene of Arthrobotrys oligospora and optimization of its prokaryotic expression conditions

为获悉少孢节丛孢菌内蒙古株Aoz1基因的详细信息,以研究捕食线虫性真菌对线虫的作用机制,本试验以捕食线虫性真菌的代表种——少孢节丛孢菌内蒙古株为研究对象,首先扩增其胞外丝氨酸蛋白酶的基因编码序列,并进行生物信息学分析,然后将其构建至原核表达载体pET-32a中,再转化至Transetta(DE3)表达感受态细胞中,进行表达条件的优化以及表达产物的纯化。结果表明,少孢节丛孢菌内蒙古株丝氨酸蛋白酶Aoz1基因与已发表的丝氨酸蛋白酶Aoz1基因的同源性为99%,其蛋白具备螺旋结构;在原核系统中进行表达,其最适IPTG诱导浓度为1.5 mmol/L,最佳诱导时间为8 h,确定了重组蛋白的最佳表达条件,并获得了重组蛋白。对该酶基因的分析,为后续有关蛋白酶和捕食线虫性真菌杀灭寄生性线虫机理方面的研究提供了参考资料。

In order to ascertain the information of the serine protease Aoz1 gene,and to study the nematode-trapping mechanism of the fungi,Arthrobotrys oligospora CHIM was selected and the serine protease Aoz1 gene was amplified and analyzed by bioinformatic software.Then the gene was connected with the prokaryotic expression vector pET32 a,and transformed into Transetta(DE3),followed by optimizing the expression conditions and purifying the expression product.The results showed that the serine protease Aoz1 gene of Arthrobotrys oligospora CHIM was 99%homologous to the published serine protease Aoz1 gene.The protein simulated by the biological software had a helical structure and was expressed successfully in the prokaryotic system.The optimal expression condi-tions of the recombinant protein were determined,such as the optimal IPTG induction concentration was 1.5 mmol/L and the optimal induction time was 8 h,and the recombinant protein was finally obtained.The bioinformatic analysis on serine protease Aoz1 gene,would be provided reference materials for the subsequent research on the protease and nematode-killing mechanism of the fungi.