| 猪肠黏膜保护因子HO-1的短小芽孢杆菌表达系统建立与分析 |
| |
| 引用本文： | 刘靖松,付京城,温丙言,杨彦宾,郭爽,焦显芹,陈瑾,黄若超,王月影,李和平.猪肠黏膜保护因子HO-1的短小芽孢杆菌表达系统建立与分析[J].中国畜牧兽医,2022,49(6):2064-2071. |
| |
| 作者姓名： | 刘靖松 付京城 温丙言 杨彦宾 郭爽 焦显芹 陈瑾 黄若超 王月影 李和平 |
| |
| 作者单位： | 1. 河南农业大学, 农业农村部动物生化与营养重点开放实验室, 郑州 450046;2. 河南农业大学动物医学院, 郑州 450046 |
| |
| 基金项目： | 河南省科技攻关计划（212102110356、212102110355） |
| |
| 摘 要： | 【目的】 对猪黏膜保护因子——血红素加氧酶-1（heme oxygenase-1，HO-1）基因进行克隆及原核表达，为研究高表达HO-1在肠黏膜损伤中的保护作用提供技术支持。【方法】 根据GenBank中公布的HO-1序列（登录号：NM_001004027.1），利用Primer Premier 6.0设计1对特异性引物，利用RT-PCR方法扩增HO-1基因片段，将其与pMD19-T克隆载体连接，转化大肠杆菌DH5α感受态细胞，筛选阳性克隆进行PCR鉴定；将载体pNCMO2与重组载体pMD19-T-HO-1进行Sal Ⅰ和Kpn Ⅰ双酶切，使用T4 DNA连接酶连接，利用电击转化技术将重组表达载体pNCMO2-HO-1转入感受态短小芽孢杆菌，使用IPTG进行诱导表达，应用SDS-PAGE和Western blotting分析HO-1在短小芽孢杆菌中的融合表达情况。【结果】 猪HO-1基因全长897 bp，编码298个氨基酸。双酶切后在约5 200和897 bp处分别观察到pNCMO2载体片段和HO-1基因片段，证明成功构建基因表达载体pNCMO2-HO-1；电转后的双酶切结果表明，在相同的位置观察到pNCMO2和HO-1片段，证明重组表达载体pNCMO2-HO-1成功导入短小芽孢杆菌。SDS-PAGE和Western blotting鉴定结果发现，在36.5 ku处出现了明显的蛋白印迹，表明成功表达了HO-1的重组蛋白，且为胞外分泌。【结论】 本研究成功构建了HO-1的重组原核表达载体，pNCMO2-HO-1重组载体可以在短小芽孢杆菌中诱导表达。
|
| 关 键 词： | 血红素加氧酶1(HO-1) 肠黏膜保护因子 短小芽孢杆菌 表达系统 重组蛋白 |
| 收稿时间： | 2021-11-08 |
Establishment and Analysis of Expression System of Porcine Intestinal Mucosal Protection Factor HO-1 in Bacillus pumilus |
| |
| LIU Jingsong,FU Jingcheng,WEN Bingyan,YANG Yanbin,GUO Shuang,JIAO Xianqin,CHEN Jin,HUANG Ruochao,WANG Yueying,LI Heping.Establishment and Analysis of Expression System of Porcine Intestinal Mucosal Protection Factor HO-1 in Bacillus pumilus[J].China Animal Husbandry & Veterinary Medicine,2022,49(6):2064-2071. |
| |
| Authors: | LIU Jingsong FU Jingcheng WEN Bingyan YANG Yanbin GUO Shuang JIAO Xianqin CHEN Jin HUANG Ruochao WANG Yueying LI Heping |
| |
| Institution: | 1. Key Laboratory of Animal Biochemistry and Nutrition of the Ministry of Agriculture and Rural Affairs, Henan Agricultural University, Zhengzhou 450046, China;2. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China |
| |
| Abstract: | 【Objective】 The purpose of this study was to clone and prokaryotic expression of heme oxygenase-1 (HO-1) gene, a protective factor of porcine mucous membrane, so as to provide technical support for studying the protective effect of highly expressed HO-1 in intestinal mucosal injury.【Method】 According to the HO-1 sequence published in GenBank (accession No:NM_001004027.1), a pair of specific primers were designed using Primer Premier 6.0, and the HO-1 gene fragment was amplified by RT-PCR, and then linked to the pMD19-T cloned vector, the recombinant vector was transformed into E.coli DH5α, and the positive clones were screened and identified by PCR.T4 DNA ligase was used to link the double digestion (Sal Ⅰ and Kpn Ⅰ) products of pNCMO2 vector and pMD19-T-HO-1 recombinant vector.The recombinant expression vector pNCMO2-HO-1 was transferred into the receptive Bacillus pumilus by electric shock transformation technology, and induced expression was performed by IPTG.The fusion expression of HO-1 in Bacillus pumilus was analyzed by SDS-PAGE and Western blotting.【Result】 The total length of porcine HO-1 gene was 897 bp, encoding 298 amino acids.The gene expression vector pNCMO2-HO-1 was double digested by restriction enzyme Sal Ⅰ and Kpn Ⅰ, the pNCMO2 vector fragment and HO-1 gene fragment were observed at 5 200 and 897 bp, respectively, which proved that the gene expression vector pNCMO2-HO-1 was successfully constructed.After electric transfer, double enzyme digestion results showed that pNCMO2 and HO-1 fragments were observed in similar locations as above, which proved that the recombinant expression vector pNCMO2-HO-1 was successfully introduced into Bacillus pumilus.SDS-PAGE and Western blotting results showed that obvious protein imprinting was appeared at 36.5 ku, indicating that HO-1 recombinant protein was successfully expressed and secreted.【Conclusion】 The recombinant prokaryotic expression vector of HO-1 was successfully constructed, and the recombinant vector of pNCMO2-HO-1 could be induced to express in Bacillus pumilus. |
| |
| Keywords: | heme oxygenase 1(HO-1) intestinal mucosal protective factor Bacillus pumilus expression system recombinant protein |
|
| 点击此处可从《中国畜牧兽医》浏览原始摘要信息 |
| 点击此处可从《中国畜牧兽医》下载免费的PDF全文 |
|
