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

根据GenBank中少孢节丛孢菌丝氨酸蛋白酶P186基因序列设计特异性引物，运用RT-PCR扩增获得目的基因片段，构建重组表达质粒pET32a-P186，转化到大肠杆菌BL21（DE3），以IPTG进行了诱导表达。结果表明，P186基因cDNA全长为1224 bp，编码407个氨基酸，其中第1～21位为信号肽序列，氨基酸序列的第156～167位、192～202位、343～353位分别是天冬氨酸（Asp160）、组氨酸（His192）、丝氨酸（Ser345）活性位点所在区域，属于Subtilase家族，包括2个潜在的N-联糖基化位点（ Asn249、Asn342）及与底物结合的S1区（ SBP） Ser251 Ile252 Gly253和Ala277 Ala278 Gly279。SDS-PAGE分析结果显示，表达产物的分子质量约为62 kDa。 Western Blot 分析结果表明，P186重组蛋白可以与抗蛋白粗提液多克隆抗体发生反应。为了揭示少孢节丛孢菌捕食线虫的分子机制，首次克隆并表达了少孢节丛孢菌丝氨酸蛋白酶P186基因，为进一步研究丝氨酸蛋白酶P186的生物学功能奠定了基础。

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 .