Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).
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.
['Pei|Zhihua|Z|', 'Bian|Lu|L|', 'Zhang|Hui|H|', 'Gao|Yunhang|Y|', 'Ma|Hongxia|H|']
D000595:Amino Acid Sequence D000818:Animals D000900:Anti-Bacterial Agents D001483:Base Sequence D003001:Cloning, Molecular D015723:Gene Library D006090:Gram-Negative Bacteria D006094:Gram-Positive Bacteria D006793:Houseflies D019476:Insect Proteins D007814:Larva D008969:Molecular Sequence Data D016133:Polymerase Chain Reaction D011994:Recombinant Proteins
cloning housefly bacteriostatic activity antibacterial protein minimum inhibitory concentration