金龟子绿僵菌(Metarhizium anisopliae HN1)几丁质酶基因的克隆及高效表达

几丁质酶是昆虫病原真菌金龟子绿僵菌致病力的主要因子之一。本实验用RT-PCR方法,从本实验室分离筛选到的高毒力金龟子绿僵菌Metarhizium anisopliae HN1中，扩增得到几丁质酶基因全长，此基因全长为1275bp，登录号为DQ011865，经Blastn分析此基因序列与M. anisopliae E6的chi1基因（AF02749）同源率为96% 。以pET-22b(+)为基础载体，构建pET-chi重组表达载体，在大肠杆菌（Escherichia. coli ）BL 21中进行表达。经SDS-PAGE分析，获得了42kDa大小的重组目的蛋白，目的蛋白占表达总蛋白含量的63.3%。菌体经冷冻与超声波破碎后，按DNS法可测得几丁质酶的活性。

Cloning chitinase gene of the entomopathogene fungus Metarhizium anisopliae HN1 and high-level expression in Escherichia . coli

Chitinases genes from Metarhizium anisopliae which is an important entomopathogenic fungus were considered one of the key factors to invade their hosts. One Metarhizium anisopliae HN1 strain was isolated and screened. A chitinase gene was amplified by RT-PCR from Metarhizium anisopliae HN1, The whole length of this gene was 1275bp,and the nucleotide sequence of the gene was 96% similarity to that of the M. anisopliae E6 accessed in GenBank ( AF02749). The gene has been registered in GenBank and its accession number is DQ011865. The gene was subcloned into prokaryon expression vector pET-22b( ), transformed this recombinant expression plasmid into E. coli strain BL 21 and effective expressed. The SDS-PAGE analysis indicated that the recombinant protein was 42kDa which is same to the reported article. The expression level of recombinant protein was about 63. 3% of whole expressed proteins , And when recombinant E. coli were crushed by freeze and supersonic wave , the activity assay indicates that the chitinase expressed in bacteria possesses biological activity.