摘要： 捕食线虫性真菌少孢节丛孢菌在侵染线虫的过程中分泌几丁质酶，为了弄清少孢节丛孢菌几丁质酶基因AO-190的分子特征和功能，对少孢节丛孢菌新疆分离株XJ-A1几丁质酶AO-190进行克隆及分子特征分析，构建原核表达载体pET32a-AO-190进行表达，并利用镍柱纯化技术纯化重组几丁质酶后，使用几丁质酶ELISA检测试剂盒测定酶活性。结果显示，XJ-A1几丁质酶AO-190基因全长1 574 bp，有4个内含子序列，序列中包含1个1 251 bp的开放阅读框（ORF），编码416个氨基酸，与少孢节丛孢菌标准株（ATCC 24927）的几丁质酶AO-190基因序列的同源性为93.33%，氨基酸序列的同源性为92.55%；有信号肽以及1个精氨酸富集区、1个双向核定位信号、1个N-糖基化位点、4个PKC磷酸化位点、6个N-酰基化位点、8个酪蛋白激酶Ⅱ磷酸化位点、1个酰胺化位点、1个低复杂度区，且含有糖苷水解酶18家族几丁质酶保守的底物结合位点SLGG和催化活性位点VDGVDLDLE，属于糖苷水解酶18家族几丁质酶；其二级结构元件有无规则卷曲、α螺旋和β折叠，三级结构为（α/β）₈圆桶形结构；系统进化分析表明，AO-190与能产生短柄黏球的Dactylellina haptotyla的几丁质酶（EPS43772.1）以及能产生收缩环的Drechslerella stenobrocha（EWC46603.1）几丁质酶亲缘关系最接近，与昆虫和脊椎动物的几丁质酶存在显著的进化距离；经SDS-PAGE和Western Blot分析，原核表达获得的重组蛋白酶分子量约为63 ku，与预期大小一致，且能与小鼠抗少孢节丛孢菌多克隆抗体发生特异性血清学反应；几丁质酶酶联免疫分析（ELISA）检测，经镍柱纯化技术纯化后的重组几丁质酶活性浓度为222 IU/L。

关键词: 少孢节丛孢菌, 几丁质酶, 原核表达, 蛋白纯化, 酶活性

Abstract: Nematode-trapping fungi can secrete chitinase in the process of infecting nematodes.In order to study the functions and molecular characteristics of chitinase, chitinase gene AO-190 of Arthrobo trys oligospora XJ-A1 isolate was cloned, analyzed and expressed in Escherichia coli by constructing prokaryotic expression vector pET32a-AO-190. The prokaryotic expression recombinant fusion protein was purified by Ni column and the chitinase activity was purified by chitinase ELISA kit.The results showed chitinase AO-190 gene had a length of 1 574 bp and four intron sequences. The sequence contained one open reading frame (ORF)with 1 251 bp that encoded 416 amino acids.The homology of the chitinase AO-190 gene sequence was 93.33% and the amino acid sequence was 92.55% respectively with Arthrobotrys oligospora standard strain (ATCC 24927)chitinase AO-190. There was a signal peptide, a Arginine-rich region profile, a Bipartite nuclear localization signal, a N-glycosylation site, a Protein kinase C phosphorylation site, a N-myristoylation site, a Casein kinase Ⅱ Amidation site and a Low-Complexity region.The proteins belonged to the family 18 glycoside hydrolase with prevalent conserved substrate binding domains SLGG and catalytic domains VDGVDLDLE.The major structural elements of secondary structure included random coils, alpha helix and extended strand while a typical (α/β)₈ phosphorylation site, a rounded bucket structure were predicted about tertiary structure.Phylogenetic analysis showed that the phylogenetic relationship of chitinase AO-190 was more close to the chitinase (EPS43772.1)originate from Dactylellina haptotyla who could produce short handle sticky ball and the chitinase (EWC46603.1) originate from Drechslerella stenobrocha who could produce constricting rings and had a significant evolutionary distance with chitinase of insects and vertebrates.The recombinant protein was identified by SDS-PAGE analysis that showed it had a molecular weight of about 63 ku, which was consistent with prediction. Western Blot analysis indicated that the recombinant protein could specifically react with polyclonal antibody against protein crude extracts.The purified recombinant chitinase activity was 222 IU/L that was measured using by chitinase ELISA kit.

Key words: Arthrobotrys oligospora, Chitinase, Prokaryotic expression, Protein purified, Chitinase activity

中图分类号: 

• Q78

• S432.4