金龟子绿僵菌(Metarhizium anisopliae HN1)几丁质酶与谷胱甘肽S-转移酶GST在大肠杆菌中的高效融合表达

High-efficient Fusion Expression of Chitinase and GST from Metarhizium anisopliae HN1 in Escheichia coli

使用RT-PCR方法,从高毒力金龟子绿僵菌Metarhizium anisopliaeHN1中,克隆得到一个全长为1 275 bp的几丁质酶基因,经Blast分析此基因序列与M.anisopliaeE6的chi1基因(AF02749)同源率为96%。将此基因克隆到pGEX-6p-1载体上,使之与载体上一个约26kD大小的谷胱甘肽S-转移酶(GST)相连,构建pGEX-chi融合表达载体,转化到大肠杆菌(Escherichia coli)BL 21中,经SDS-PAGE结果分析显示:表达出的融合蛋白大小为68 kD,此目的蛋白占表达总量的64.5%。经破碎处理后可检测到几丁质酶活性。

Chitinase genes from Metarhizium anisopliae that is an important entomopathogenic fungus were eonsidered one of the key factors to invade their hosts. Total RNA was extracted from Metarhizium anisopliae HNI strain and chitinase gene was amplified by RT-PCR. The whole length of this gene was 1 275 bp, and the nucleotide sequence of the gene was 96% similarity to that of the M. anisopliae D5（AF02749）.Then the gene was subcloned into prokanyo expression vector pGEX-6p-land connected with GST.The clones were identified by enzyme digestion and sequenced. This expression plasmid was transformed into E. coli strain BL 21 and effective fusion was expressed. SDS-PAGE analysis indicated that the recombinant fusion protein was 68 kD. The level of expression fusion protein was about 64.5% of total expressed proteins. The activity of chitinase could be detected after fragmentation treatment.