重组Gallinacin-3的克隆及在大肠杆菌中的表达  

Cloning and Expression of Gallinacin-3 in Escherichia coli

作　　者：赵建增 乔彦良[1] 李朝阳[1] 

机构地区：[1]山东信得药业有限责任公司,山东青岛266701

出　　处：《中国兽药杂志》2006年第2期1-4,34,共5页Chinese Journal of Veterinary Drug

基　　金：山东省科学技术发展项目031030101

摘　　要：防御素是昆虫、植物和动物体内抗感染及激发特异性抗感染免疫的重要抗菌肽。Gallinacin-3(gal-3)可能是鸡体内最重要的β-防御素,但它的功能还未得到研究证实。本试验从鸡黏膜表皮组织中克隆了gal-3编码基因,并在大肠杆菌中成功表达了gal-3与谷胱甘肽转硫酶(GST)融合蛋白。经IPTG诱导后,融合蛋白的产量占细菌总蛋白的33.6%左右,主要以包涵体形式存在。用亲合层析技术从大肠杆菌裂解液中纯化出可溶性重组蛋白,并用酶切除目标蛋白的融合伴侣。抑菌试验发现,重组gal-3-GST融合蛋白在未去除融合伴侣时,就与游离的gal-3有着相同的抑菌活性,对沙门氏肠炎杆菌和志贺氏痢疾杆菌的最小杀菌浓度在20μg/mL(相当于约4μg/mL纯gal-3)左右,与其他物种β-防御素的活性相当。Defensins are a group of endogenous antimicrobial peptides that plays indispensable functions in triggering adaptive immune responses. Due to its wide in vivo distribution, gallinacin-3 might be the most important defensin in chickens. However, its biological importance remains uninvestigated. In this study, the gene encoding for Gallinacin-3 was isolated from chicken＇ s tongue epithelia by using reverse transcriptation polymerase chain reaction. It was stably expressed in Escherichia coli when being fused under the gene for glutathione S-transferase. Following induction with IPTG, the recombinant protein was 33.6% of the total bacterial proteins, most of which was in cytorrhyctes form. The soluble fusion protein, prepared from E. coli extracts by affinity chroma-tography, could suppress the growth of Salmonella enteritidis and Shigella shigae. The minimal bactericidal concentration was less than 20μg/mL, equaling to about 4 μg/mL native gal-3.

关 键 词：鸡 Β-防御素 大肠杆菌 融合蛋白 抑菌作用 

分 类 号：Q786[生物学—分子生物学]