The Prokaryotic Expression And Activities Assay Of Differential Gene Md-FBPâ… , Md-CBPâ… and Md-LSZâ… from Musca Domestica

Antibiotics are the most commonly used anti-infective drugs.But the the drug resistance of bacteria has become more and more serious since the abuse of antibiotics in clinical application. Therefore, it is urgent to develop a new broad-spectrum and no-drug resistance alternative medication for antibiotic,which includes antibiotic peptides. Antibiotic peptides not only originated from flora and fauna, but also from insects in nature.the anti-bacteria bio-active substances from Musca domestica autoimmunity have the characteristics of low toxicity, no drug-resistant, and etc, which has attracted widespread attention of researchers both domestic and overseas. Therefore, the research into Musca domestica antibiotic peptides and its immune response mechanism by using gene engineering technology has become the current hotspot.This research was based on the genes screened from suppression subtractive libraries（SSH） in Musca domestica larvae induced by Pasteurella multocida and Salmonella. The three differential genes were amplificated by RACE-PCR techniques. The amplification products were sequenced and analyzed by both BLAST and biology analysis. Two Full-length PCR products（Md-CBPâ…  and Md-LSZⅠ） were cloned into pMD18-T Vector by T-A cloning technique. The positive cloned plasmids which are pMD18-T-Md-LSZâ… and pMD18-T-Md-CBPâ… have been constructed successfully. The Md-LSZâ…  and Md-CBPâ…  genes were subcloned into pET-32a（+） prokaryotic expression vector to construct recombinant expression plasmids. The recombinant expression plasmids have been transformed into E.coli BL21（DE3） or E.coli Transetta（DE3） and highly expressed. the fusion protein was purified,futher more, the binding affinity and antibacterial activity was analysed, the main results are as follows :（1） The amplication of three differential genes were made by using RACE-PCR technique, the full-length ORF sequences respectively are 303 bp,429bp,366 bp.BLAST analysis showed that Md-LSZâ… has 98% similarity with the lysozyme 1-like gene which GenBank accession number is XP₀11292185.1（the 44,46 and 73 position are different）.Md-CBPâ… has 98% similarity with the larval cuticle protein 9-like gene which GenBank accession number is XP₀11296921.1（the 89 position is different）.Md-FBPâ… has 99% similarity with the fructose-diphosphate aldolase isoform X2 gene which GenBank accession number is XP₀11297661.1（the 68 position is different）.Biological information analysis of two genes which are Md-LSZâ… and Md-CBPâ… indicates that Md-LSZâ…  gene encodes a 143 amino acids hydrophobic protein. its theoretical isoelectric point is 5.22. Bio-active site prediction showed that the 73 amino acid locates in Casein kinase II phosphorylation site. Md-CBPâ… gene encodes a 122 amino acids hydrophilic protein. its theoretical isoelectric point is 5.63, conservative region prediction showed that 89 amino acid located in conserved motif of Md-CBPâ… . The results of secondary structure prediction show that the main structure of both genes encoded proteins are β-sheet, alpha helix, irregular coil and β-turn.（2） The prokaryotic recombination expression plasmids of pET-32a-Md-LSZâ…  and pET-32a-Md-CBP â… were constructed successfully and also highly expressed in E.Coli. Therein, the fusion protein of Md-LSZ is inclusionâ… -body protein, the other is soluble protein. Fusion proteins with single band was obtained by purification.（3） Further more, the preliminary study was made on the affinity of pET-32aMd-CBPâ… fusion protein which revealed that the fusion protein has binding affinity of both chitin and cellulose, and the affinity of chitin was stronger than cellulose.（4） The antibacterial experimental results show that Md-LSZâ… fusion protein selectively inhibit the growth of salmonella but not inhibit Escherichia coli or Staphylococcus aureus. Md-CBPâ… fusion protein did not have bacteriostatic action on these three pathogens.These results have lay the foundation for the further study on the mechanism of these two fusion proteins, and also provide a basis for the study on both the function of Musca domestica antibiotic peptides and the related immune mechanism.