The Expression And Activity Of Different Expressed Genes Of Musca Domestica Larvae Before And After Stimulated With Pathogens

Musca domestica larvae live in a wet, organic matter-enriched environment. They often carry large amounts of pathogens and harmful factors, but they rarely show signs of disease. The researchers predict that the unique immune defenses of Musca domestica are largely due to the disease resistance substances produced by their bodies. The disease resistance substances not only have the broad-spectrum antimicrobial ability, but also have the activity of anti-fungi, anti-protozoa, anti-viruses, and even kill cancer cells without damaging normal cells in the body, therefore, the disease resistance substances has become the focus of scholars. Musca domestica larvae can synthetise a variety of disease resistance substances increment expression after stimulated with pathogens, such as antimicrobial peptides, lections, lysozyme, in vitro larval secretions, chitin shell glycans, and some unknown factors. The discovery of antimicrobial substances makes it possible that new medicines replace antibiotics and thus alleviate the antibiotic-resistant pressure of bacteria.In this study, three different expressed genes from Musca domestica larvae stimulated with Bovine Pasteurella multocida, Mycoplasma hyopneumoniae and chicken pathogenic E. coli, respectively, were studied. The genes were cloned and expressed, the activity of the expressed products were also researched. An uncharacterized gene named MdU-F501 from suppression subtractive library(SSH) in Musca domestica larvae stimulated with Bovine Pasteurella multocida was amplificated by RACE-PCR techniques; the open reading frame(ORF) product was sequenced and analyzed. Full-length PCR product was connected with the prokaryotic expression vector pGEX-4T-1 and the recombinant expression plasmid MdU-F501-pGEX-4T-1 was constructed successfully. The uncharacterized gene MdU-132 from suppression subtractive library(SSH) in Musca domestica larvae stimulated with Mycoplasma hyopneumoniae and the MdL-II from suppression subtractive library(SSH) in Musca domestica larvae stimulated with chicken pathogenic E. coli were sub-cloned into pET-32a(+) to construct recombinant expression plasmids MdU-132-pET-32 a and MdLII-pET-32 a. They were transformed into E.coli BL21(DE3) and induced by IPTG, then, the expressed results were detected by SDS-PAGE electrophoresis. The fusion protein was purified by affinity chromatography. The antibacterial activity of the purified proteins was tested by Oxford cup method. The main results are as follows:(1)The full-length ORF of MdU-F501 sequence is 525 bp. Its theoretical isoelectric point is 8.23, without transmembrane region. It is not a membrane protein or secreted protein, a signal peptide region has been found throughout the protein by bioinformatics analysis.(2)The recombinant expression plasmid MdU-F501-pGEX-4T-1、MdLII-pET-32 a and MdU-132-pET-32 a were constructed successfully. They were expressed in E.coli BL21(DE3). The best conditions for MdLII-pET-32 a fusion protein expression were IPTG concentration 0.6mM, the induction temperature is 37℃.(3)The high purity and the purified fusion protein showed a single band with affinity chromatography. The antibacterial activity detection of three fusion proteins showed that the fusion protein GST-MdU-F501 had no antibacterial activity. Fusion protein Trx-MdLII have the inhibitory effect on Streptococcus suis resistant strains, Escherichia coli resistant strains and Pasteurella multocida bovine resistant strains, but no inhibitory effect on Mycoplasma hyopneumoniae. The fusion protein Trx-MdU-132 haved inhibitory effect on Streptococcus suis resistant strains and Escherichia coli resistant strains, there was a slight inhibitory effect on Mycoplasma hyopneumoniae.