华北农学报Issue(4)：93-97,5.
少孢节丛孢菌胞外丝氨酸蛋白酶 P186基因的克隆、序列分析及表达
Cloning and Prokaryotic Expression of Serine Protease P186 Gene of Arthrobotrys oligospora
摘要
Abstract
A pair of specific primers derived from Arthrobotrys oligospora Serine Protease P186 gene in Gen-Bank was designed and used to amplify P186 gene by RT-PCR from total RNA extracted from Arthrobotrys oligospo-ra.The RT-PCR product was cloned into the expression vector pET 32a to generate,recombinant pET32a-P186,and pET32a-P186 plasmid was transformed into E.coli BL21(DE3)and induced by IPTG.Sequence analysis demon-strated that P186 cDNA contained a insert of 1 224 bp encoding 407 amino acids,Among them 1-21 for the signal peptide sequence,amino acid sequence of 156-167,192-202,343-353 were aspartic acid(Asp160),histidine (His192)and serine(Ser345)active site area,respectively,which are belonging to the family of Subtilase.The amino acid also contained two potential N-terminal glycosyauon sites ( Asn249 ,Asn342 ) and a substrate binding region of S 1 ( SBP) Ser251 Ile252 Gly253 and Ala277 Ala278 Gly279 .SDS-PAGE analysis showed that the product had a molecular weight of about 62 kDa.Western Blot analysis indicated that the recombinant protein could specifically react with poly-clonal antibody against protein crude extracts .In order to reveal the molecular mechanism of Arthrobotrys oligospora predatory nematodes,In this study,the serine protease P186 gene of Arthrobotrys oligospora was firstly cloned and expressed ,which laid a foundation of further study on biological function of P 186 .关键词
少孢节丛孢菌/丝氨酸蛋白酶P186/克隆/表达Key words
Arthrobotrys oligospora/Serine protease P186/Cloning/Expression分类
生物科学引用本文复制引用
赵海龙,孟庆玲,乔军,陈双庆,王国超,谢堃,胡政香,才学鹏..少孢节丛孢菌胞外丝氨酸蛋白酶 P186基因的克隆、序列分析及表达[J].华北农学报,2014,(4):93-97,5.基金项目
国家农业公益行业专项子课题（201303037-5）；国家自然科学基金项目（31260601）；兵团博士资金项目 （）
