家蝇幼虫抗菌肽天蚕素基因的克隆及其在大肠杆菌中融合表达(英文)  

Cloning of the antibacterial peptide cecropin gene of Musca domestica larvae and its fusion expression in Escherichia coli

作　　者：徐建华[1] 朱家勇[2] 金小宝[2] 许琴英[3] 刘雷山[2] 马艳[2] 王艳[2] 

机构地区：[1]广州中医药大学第二附属医院(广东省中医院)检验科,广州510120 [2]广东药学院基础医学院,广州510224 [3]广东医学院病原生物学教研室,湛江524023

出　　处：《中国人兽共患病学报》2007年第4期311-318,共8页Chinese Journal of Zoonoses

基　　金：grant of State Natural Science Foundation(30671832);Key Science and Technology Programof Guangdong Province (2003B31602);Key Science and Technology Program of Guangzhou (2005Z3-E0211) .

摘　　要：目的从家蝇三龄幼虫中提取总RNA,先利用RT-PCR扩增编码天蚕素Cecropin的cDNA序列,克隆入T载体pUCm-T并测定其序列。然后以pUCm-T/Cecropin为模板,通过PCR方法扩增Cecropin成熟肽cDNA序列,并将该序列的N-端大肠杆菌稀有密码子GGA点突变为GGC,C-端终止密码子TAA前加进一个天冬酰胺(Asn)密码子AAC,命名为mCecropin。mCecropin基因序列克隆至融合表达载体pGEX-4T-1中。酶切分析和测序鉴定后,将阳性重组子质粒转入不同的大肠杆菌宿主细胞中进行融合表达。经SDS-PAGE分析,选用E.coliBL21(DE3)作为表达宿主菌。表达的融合蛋白GST-mCecropin通过GSTrap亲合柱纯化后用凝血酶酶切GST标签,mCecropin经HiTrap柱进一步纯化后,具有较强的抗菌活性。In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the eDNA sequence encoding the ORF of cecropins was amplified by RT-PCR, and the target fragment was further sequenced after being cloned into T vector pUCm-T. Then, the eDNA sequence of the mature cecropins was amplified by PCR with recombinant plasmid pUCm-T/cecropin as template, the N-terminal rare codon GGA of E. coli was changed to the favorable codon GGC, and a Asn codon AAC was added in front of the stop coden TAA in the C- terminus. This mutant gene designated as mCecropin was then ligated with the fusion expression vector pGEX-4T-1. After restriction analysis and DNA sequencing, the positive recombinant plasmid pGEX-4T-1/mCecropin was transformed to different strains of E. coli cells and the fusion protein was ex- pressed after IPTG induction. The fusion protein was assayed by SDS-PAGE and the E. coli BL21 （DE3） cell was chosen as the host cell for the expression of the fusion protein. The expressed fusion protein GST-mCecropin was purified by GSTrap affinity coloum and the GST marker was then cleaved by thrombin. In this way, the fusion protein mCecropin with antibacterial activity was obtained after purification with HiTrap benzamidine column.

关 键 词：家蝇 天蚕素 抗菌肽 基因克隆 融合表达 

分 类 号：R384.2[医药卫生—医学寄生虫学]