Cloning of the antibacterial peptide cecropin gene of Musca domestica larvae and its fusion expression in Escherichia coli
10.3969/j.issn.1002-2694.2007.04.001
- VernacularTitle: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
- Author:
Jianhua XU
;
Jiayong ZHU
;
Xiaobao JIN
;
Qinying XU
;
Leishan LIU
;
Yan MA
;
Yan WANG
- Publication Type:Journal Article
- Keywords:
Musca domestica;
cecropin;
antibacterial peptide;
gene cloning;
fusion expression
- From:
Chinese Journal of Zoonoses
2007;(4):311-318
- CountryChina
- Language:Chinese
-
Abstract:
In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the cDNA 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 cDNA 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 expressed 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.
