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

Abstract

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

CLC Number:

S852.4

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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Establishment and Analysis of Expression System of Porcine Intestinal Mucosal Protection Factor HO-1 in Bacillus pumilus

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