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中国精品科技期刊2020
何雨峰, 韦胜, 岳硕豪, 徐泽, 秦松, 黄莹琪, 邓颖, 王伟平. 红曲菌Mn-SOD基因克隆、表达及序列分析[J]. 食品工业科技, 2020, 41(18): 81-86. DOI: 10.13386/j.issn1002-0306.2020.18.013
引用本文: 何雨峰, 韦胜, 岳硕豪, 徐泽, 秦松, 黄莹琪, 邓颖, 王伟平. 红曲菌Mn-SOD基因克隆、表达及序列分析[J]. 食品工业科技, 2020, 41(18): 81-86. DOI: 10.13386/j.issn1002-0306.2020.18.013
HE Yu-feng, WEI Sheng, YUE Shuo-hao, XU Ze, QIN Song, HUANG Ying-qi, DENG Ying, WANG Wei-ping. Cloning,Expression and Sequence Analysis of Mn-SOD Gene of Monascus[J]. Science and Technology of Food Industry, 2020, 41(18): 81-86. DOI: 10.13386/j.issn1002-0306.2020.18.013
Citation: HE Yu-feng, WEI Sheng, YUE Shuo-hao, XU Ze, QIN Song, HUANG Ying-qi, DENG Ying, WANG Wei-ping. Cloning,Expression and Sequence Analysis of Mn-SOD Gene of Monascus[J]. Science and Technology of Food Industry, 2020, 41(18): 81-86. DOI: 10.13386/j.issn1002-0306.2020.18.013

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

  • 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EcoR Ⅰ和Hind Ⅲ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。

     

    Abstract: 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 EcoR Ⅰ and Hind Ⅲ,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.

     

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