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当前位置: 首页 > 文章 > 少孢节丛孢菌新疆分离株几丁质酶AO-14基因的克隆表达 畜牧与兽医 2017,49 (11) 118-123
Position: Home > Articles > Cloning and expression of chitinase AO-14 gene from Arthrobotrys oligospora XJ-A1 Animal Husbandry & Veterinary Medicine 2017,49 (11) 118-123

少孢节丛孢菌新疆分离株几丁质酶AO-14基因的克隆表达

作  者：
钟文强;孟庆玲;乔军;陈英;贡莎莎;才学鹏
单  位：
石河子大学科技学院;中国农业科学院兰州兽医研究所
关键词：
少孢节丛孢菌;几丁质酶;AO-14基因;克隆;序列分析;原核表达
摘  要：
利用RT-PCR扩增获得少孢节丛孢菌新疆分离株XJ-A1几丁质酶AO-14目的基因片段,对该基因进行克隆和分子特征分析;构建重组表达质粒p ET32a-AO14,转化到大肠杆菌进行诱导表达,用SDS-PAGE和Western blot分析表达的重组蛋白。结果表明,AO-14基因c DNA全长为1 200 bp,编码399个氨基酸;编码蛋白几丁质酶AO-14属于糖苷水解酶18家族,具有典型的几丁质酶催化区保守序列SXGG和DGXDXDWE,氨基酸序列的第131~139为活性位点所在区域,无信号肽序列。SDS-PAGE分析结果显示,AO-14表达产物的分子质量约为60 ku;Western-blot分析结果表明,重组蛋白可以与抗少孢节丛孢菌多克隆抗体发生反应,证实在大肠杆菌中实现了少孢节丛孢菌XJ-A1几丁质酶AO-14基因的表达,为进一步研究少孢节丛孢菌几丁质酶AO-14生物学功能奠定了基础。
译  名：
Cloning and expression of chitinase AO-14 gene from Arthrobotrys oligospora XJ-A1
作  者：
ZHONG Wenqiang;MENG Qingling;QIAO Jun;CHEN Ying;GONG Shasha;CAI Xuepeng;Department of Animal Science and Technology,Shihezi University;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences;
单  位：
Department of Animal Science and Technology,Shihezi University%Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences
关键词：
Arthrobotrys oligospora;;chitinase;;AO-14 gene;;cloning;;sequence analysis;;prokaryotic expression
摘  要：
The target gene fragment of chitinase AO-14 of Arthrobotrys oligospora XJ-A1 was obtained by RT-PCR.The gene was cloned and its molecular characterizations were analyzed.The recombinant plasmid p ET32 a-AO14 was transformed into Escherichia coli BL21 and induced by IPTG.The recombinant protein was identified by SDS-PAGE and Western blot.The results showed that the full length of AO-14 gene was 1200 bp,encoding 399 amino acids.Chitinase AO-14 belongs to the family of glycoside hydrolase 18,which is characterized by a typical chitinase catalytic region with the conserved sequence SXGGDGXDXDWE,amino acid sequence of 131 to 139 as the active site area and no signal peptide sequence.SDS-PAGE analysis showed that the protein product had a molecular weight of about 60 ku.Western blot analysis indicated that the AO-14 recombinant protein could specifically react with polyclonal antibody against protein crude extracts.This study provides a basis for investigation of chitinase AO-14 function.

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