title: Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva.
abstract: Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram- bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, PR China.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Pei
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, PR China.
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Sun
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Tang
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, PR China.
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, PR China.
authors.forename: Yunhang
authors.initials: Y
authors.lastname: Gao
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, PR China. Electronic address: hongxia0731001@163.com.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Ma
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D003001:Cloning, Molecular; D015723:Gene Library; D006090:Gram-Negative Bacteria / Q000145:classification; D006793:Houseflies / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000382:microbiology; D019476:Insect Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D007814:Larva / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins
keywords: Antimicrobial peptide; Bacteriostatic activity; Cloning; Musca domestica