抗菌肽Cecropin A基因原核表达及表达产物的鉴定(英文)  

Prokaryotic expression of antibiotic peptide Cecropin A gene and identification of expression products

作　　者：陈磊[1] 

机构地区：[1]河南中医学院第一附属医院心内科,河南省郑州市450000

出　　处：《中国组织工程研究与临床康复》2011年第24期4549-4552,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:Cecropins是一种具有抗菌活性的小分子蛋白质。采用真核细胞表达或人工合成Cecropins,效率低、成本高。目的:克隆表达家蝇抗菌肽基因Cecropin A,并对其重组表达产物进行鉴定。方法:依据GenBank中家蝇Cecropin A基因序列设计特异性引物,用RT-PCR从家蝇幼虫组织中扩增Cecropin A成熟肽基因,将其克隆入原核表达载体pET32a中,与表达载体中的Thioredoxin基因构成融合基因,并转化E.coli BL21。经异丙基-β-D硫代半乳糖苷诱导表达。采用家蝇幼虫血淋巴免疫家兔获得抗血清;Western blot和三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定诱导的重组蛋白质。结果与结论:经异丙基-β-D硫代半乳糖苷诱导,E.coliBL21可表达Cecropin A成熟肽,兔抗家蝇幼虫血清、三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及Western blot结果证实表达产物为Cecropin A成熟肽。说明使用原核表达系统可以获得天然Cecropin A成熟肽。BACKGROUND：Cecropins are a kind of micromolecule protein with antibacterial activity.Eukaryotic cell-expressed or artificially synthesized Cecropins is characterized by low efficiency and high cost.OBJECTIVE：To clone and express an antibacterial peptide gene of Musca domestica Cecropin A,and to identify recombinant expression product.METHODS：Mature Musca domestica Cecropin A encoding nucleotide sequence was searched from the GenBank and amplified by RT-PCR.The gene of Musca domestica Cecropin A was cloned into prokaryotic expression vector pET32a and fused with gene of Thioredoxin（Trx） and expressed in E.coli BL2l（DE3）.After induction by isopropyl-β-D-thiogalactoside,the sera of the immunized rabbits were collected after rabbits were immunized with the hemolymph of housefly larvae.Recombinant protein was identified by western blot analysis and N-[Tris（hydroxymethyl）methyl]glycine-sodium dodecylsulfate-polyacrylamide gel electrophoresis.RESULTS AND CONCLUSION：After induction by isopropyl-β-D-thiogalactoside,E.coli BL21 expressed mature Cecropin.Rabbit anti-housefly larvae sera,N-[Tris（hydroxymethyl）methyl]glycine-sodium dodecylsulfate-polyacrylamide gel electrophoresis and western blot analysis results confirmed that expression products were mature Cecropin.These suggest that prokaryotic expression system can be utilized to obtain natural mature Cecropin.

关 键 词：家蝇 CECROPIN A 多克隆抗体 融合表达 三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 

分 类 号：R318[医药卫生—生物医学工程]