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

doi:10.16431/j.cnki.1671-7236.2022.06.006

摘要/Abstract

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

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.

Key words: heme oxygenase 1(HO-1); intestinal mucosal protective factor; Bacillus pumilus; expression system; recombinant protein

中图分类号:

S852.4

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.

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 and Veterinary Medicine, 2022, 49(6): 2064-2071.

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