红曲菌Mn-SOD基因克隆、表达及序列分析  

Cloning,Expression and Sequence Analysis of Mn-SOD Gene of Monascus

作　　者：何雨峰 韦胜 岳硕豪 徐泽 秦松 黄莹琪 邓颖 王伟平 HE Yu-feng;WEI Sheng;YUE Shuo-hao;XU Ze;QIN Song;HUANG Ying-qi;DENG Ying;WANG Wei-ping(Key Laboratory of Fermentation Engineering,Ministry of Education,College of Bioengineering and Food,Hubei University of Technology,Wuhan 430068,China;Center of Applied Biotechnology,Wuhan Bioengineering Institute,Wuhan 430415,China)

机构地区：[1]湖北工业大学生物工程与食品学院发酵工程教育部重点实验室,湖北武汉430068 [2]武汉生物工程学院应用生物技术研究中心,湖北武汉430415

出　　处：《食品工业科技》2020年第18期81-86,共6页Science and Technology of Food Industry

基　　金：国家自然科学基金(31101349);湖北工业大学2018年省级大学生创新创业训练计划培育项目(201810500060)。

摘　　要：为得到产Mn-SOD工程菌及表达产物,通过设计出红曲菌H4000 Mn-SOD简并引物得到目的基因片段,再设计全长引物利用PCR技术扩增红曲霉Mn-SOD基因的全长序列。经EcoRⅠ和HindⅢ酶切后连接至相同酶切的表达载体pET28a,并转化至E.coli BL21进行诱导表达。克隆得到的基因预测编码152个氨基酸,预测相对分子量为17 kDa。同时,将克隆得到的Mn-SOD基因与NCBI数据库进行比对,发现该序列与橙色红曲霉超氧化物歧化酶(SOD)基因相似度达到99%,与炭疽菌、米曲霉、黄曲霉的Mn-SOD基因也有较高的相似度。通过SDS-PAGE检测蛋白表达情况,目标蛋白相对分子质量约为19 kDa,与预测分子量基本相符。该表达蛋白在pH2.0保温处理1 h,仍具有较高的Mn-SOD酶活。In order to obtain the engineering bacteria and expression products of Mn-SOD in Monascus,the target gene fragment was obtained by designing the degeneracy primers of Monascus H4000,and then the full-length primers were designed to amplify the full-length sequence of Mn-SOD gene of Monascus by PCR.The target gene was digested by Eco RⅠand Hin dⅢ,and then linked to pET28a which was digested by the same enzyme,and then transformed into E.coli BL21 to induce expression.The obtained gene was predicted to encode 152 amino acids with a predicted relative molecular weight of 17 kDa.The cloned gene of Mn-SOD was compared with the NCBI database,it was found that the similarity of the sequence was 99%with that of sod gene of Monascus orange,and the sequence had higher similarity with the Mn-SOD gene of Anthracis,Aspergillus oryzae and the Aspergillus flavus.The molecular weight of Mn-SOD was estimated to be 19 kDa by SDS-PAGE.The expressed protein showed good acid resistance,which still had enzyme activity at 1 h after pH2.0 treatment.

关 键 词：红曲菌 锰超氧化物歧化酶(Mn-SOD) 基因克隆 序列分析 诱导表达 

分 类 号：TS201.3[轻工技术与工程—食品科学]