解淀粉芽孢杆菌ASR-12抗菌蛋白的分离纯化及其编码基因克隆  

Isolation and purification of antimicrobial protein from Bacillus amyloliquefaciens ASR-12 and cloning of its coding gene

作　　者：王彪 何鹏飞[1] 王再强[1] 吴毅歆[2,3] 何朋杰 崔文艳 何月秋[2,3] WANG Biao;HE Pengfei;WANG Zaiqiang;WU Yixin;H E Pengjie;CUI Wenyan;HE Yueqiu(College of Plant Protection,Yunnan Agricultural University,Kunming 650201;College of Agronomy and Biotechnology,YAU,Kunming 650201;National and Local Joint Engineering Research Center for Screening and Application of Microbial Strains,Kunming 650217)

机构地区：[1]云南农业大学植物保护学院,昆明650201 [2]云南农业大学农学与生物技术学院,昆明650201 [3]微生物菌种筛选与应用国家地方联合工程研究中心,昆明650217

出　　处：《安徽农业大学学报》2020年第2期299-303,共5页Journal of Anhui Agricultural University

基　　金：科技部“十三五”重点研发计划项目(2018YFD0201200);云南省自然科学基金(2017FB051)共同资助。

摘　　要：为了从菌株ASR-^12发酵液中分离纯化获得抗菌蛋白。将菌株ASR-^12发酵液经硫酸铵沉淀、透析除盐后获得粗蛋白,粗蛋白进行热处理和Q Sepharose Fast Flow柱层析,纯化后的蛋白进一步进行LC-MS鉴定,并克隆其编码基因进行测序。粗蛋白经热处理和柱层析后得到5个吸收峰,活性检测结果显示峰Ⅳ具有较高抑菌活性,经SDS-PAGE检测显示为单一条带,分子量约为50k Da。LC-MS鉴定结果显示抗菌蛋白与M42家族肽酶同源性最高,覆盖度达到80%;并能从菌株ASR-^12基因组中克隆到M42家族肽酶编码基因,测序结果经BLAST比对与解淀粉芽孢杆菌B9601-Y2的M42家族肽酶编码基因同源性达100%。菌株ASR-^12产生的抗菌蛋白中,M42家族肽酶为主要活性成分。In order to isolate and purify the antibacterial protein from fermentation broth of Bacillus amyloliquefaciens ASR-^12. Fermentation broth from strain ASR-^12 was precipitated with ammonium sulfate and desalted by dialysis to obtain the crude protein. The crude protein was subjected to heat treatment and Q Sepharose Fast Flow column chromatography. The purified protein was further identified by LC-MS and its coding gene was cloned for sequencing. 5 absorption peaks were obtained after heat treatment and column chromatography, and active detection results showed that the peak Ⅳ has a higher antagonistic activity, which displayed a single band with about 50 KDa by SDS-PAGE detection.The results of LC-MS identification showed that the antibacterial protein had the highest homology with M42 family peptidase and the coverage reached 80%. The gene encoding M42 family peptidase was cloned from the genome of strain ASR-^12. The sequencing results after BLAST alignment showed that their homology reached 100% comparing with M42 family peptidase encoding gene from bacillus amyloliquefaciens B9601-Y2. M42 family peptidase is the main active component in the antibacterial protein produced by strain ASR-^12.

关 键 词：硫酸铵沉淀 分离纯化 抑菌活性 基因克隆 

分 类 号：S476[农业科学—农业昆虫与害虫防治] S482.292[农业科学—植物保护]