title: Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).
abstract: Musca domestica L. (Diptera: Muscidae), the housefly, exhibits unique immune defenses and can produce antibacterial substances upon stimulation with bacteria. On the basis of the cDNA library constructed using the suppression subtractive hybridization method, a 1188-bp antibacterial substance gene, which we named AS566, was amplified by rapid amplification of cDNA ends from M. domestica larva stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In this study, the full-length AS566 gene was cloned and inserted into a His-tagged Escherichia coli (Enterobacteriaceae: Escherichia) prokaryotic expression system to enable production of the recombinant protein. The recombinant AS566 protein was purified in denatured form from inclusion bodies and renatured to obtain functionally active AS566 protein. The bacteriostatic activity of the recombinant purified AS566 protein was assessed using the Oxford plate assay system and the results indicated that AS566 had antibacterial activity against six bacteria, including an E. coli clinical isolate, S. pullorum, Streptococcus bovis (Streptococcaceae: Streptococcus), Streptococcus suis, and Staphylococcus aureus (Staphylococcaceae: Staphylococcus) in vitro. The antibacterial activity of AS566 toward Gram- bacteria was two times greater than that against Gram+ bacteria. The sequencing results and BLAST analysis showed that the antibacterial substance gene AS566 was not homologous to any other antibacterial substance genes in GenBank. The antibacterial mechanisms of the newly discovered AS566 protein warrant further study.
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, People's Republic of China.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Pei
authors.affiliation: Liaoning Province Agricultural Economic School, People's Street No. 14, Guta District, Jinzhou 121000, Liaoning Province, People's Republic of China.
authors.forename: Lu
authors.initials: L
authors.lastname: Bian
authors.affiliation: Animal Husbandry Bureau of Yongji County, Tianjin Street, Yongji County, Jilin 132100, Jilin Province, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No. 2888, Changchun 130118, People's Republic of 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, People's Republic of China hongxia0731001@163.com.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D006090:Gram-Negative Bacteria / Q000187:drug effects*; D006094:Gram-Positive Bacteria / Q000187:drug effects*; D006793:Houseflies / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D019476:Insect Proteins; D011994:Recombinant Proteins
keywords: antibacterial protein; bacteriostatic activity; cloning; housefly; minimum inhibitory concentration