| Antibacterial peptides(ABP) are small kinds of bioactive peptides in hameolymph encoded by some structural genes,and are widespread in the natural organisms, which are an important composition of its immune system, They have non-specific resistance on bacteria, fungi, viruses, parasites and other pathogens, at the same time, These peptides can inhibit the growth of tumor cell and multi-drug resistant bacteria without harmless on the normal cell organisms. Their mechanism is unique so that they have huge potential for the development of new antimicrobial agents. For that reasons, antibacterial peptides have increasingly become a hotspot in the area of life sciences research.From larvae to adult, Musca domestica lives in the landfill, livestock manure ,where various pathogens reproduce, and can bring a variety of harmful pathogens to human and livestock without being infected and death strangely. This may have originated from its unique natural immune substances to combat various microorganisms, in which ABP play a important role. Musca domestica in China is a good insect resource in the development and application of the medicine because of their wide distributation, easy being raised and lower cost of breeding. Natural antibacterial peptides extracted from the Housefly body costs highly of material, and the way of synthesis are also very expensive. Through genetic engineering methods may be an ideal way to prepare ABP. But this method is on the basis of the obtaining of the related antimicrobial peptide genes.As a new molecular biology technology ,the gene chip technology was developed in the field of life science in recent years,which has the fast, efficient, sensitive, parallel and many other advantages. Unlike the traditional method of studies by which only one or few genes has been studied, gene chip technology has a very good application prospect on screening in differential gene expression,discoverying the novel genes, detecting the gene of mutations and analyzing the gene polymorphism. Using this technology for the study of screening Musca domestica antibacterial peptide gene has not been reported. In this paper, The oligonucleotide hybrid probes were designed according to the part gene sequences in GenBank . Then, oligonucleotide (oligo) chips were constructed to screen the candidate genes associated with Musca domestica antibacterial peptides, and cloned and analyzed a novel antibacterial peptide gene of Musca domestica.ObjectiveTo screen the candidate genes associated with Musca domestica antibacterial peptides using gene chip technology, clone and analyze a novel antibacterial peptide gene of Musca domestica,which lay a solide foundation for the further research the Musca domestica antimicrobial peptide structure and functions, reveal their role in their innate immune system ,and then , prepare the Musca domestica antibacterial peptide through genetic engineering methods.Methods1. constructed a antibacterial peptide gene chip and screened the antibacterial peptide associated genes of Musca domestica Part insect antibacterial peptide gene orders were acquired from the GenBank, by analyzing mRNA sequence of those insect antibacterial peptide genes with associated free biology softwares in National Center for Biotechnology Informational ( NCBI ) , the encoded areas and the conservative domains of those antibacterial peptide genes were found, Then,the oligo software of Array Designer 2.0 was applied to select hybrid probes with high specificity, identical length and similar melting temperature(Tm) from the conservative sequences, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter , those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by E.coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analysised using the software of MIDAS.2.cloned and analyzed a novel antibacterial peptide gene of Musca domesticaA pair of degenerate primers were designed according the conservative domains in the amino acid orders of the insect antibacterial peptide gene Diptericin,which had been screened with gene chips in the first part, a homologous fragment was amplified by RT-PCR. A specific 5′primer was designed from the cloned homologous fragment ,combined the 3′primer in the plasmid ofλTripEx2 of the cDNA library of Musca domestic larve( which had been constructed by our laboratory),with the template of cDNA library, the 3′end cDNA sequence was amplified by PCR. With the same principle,a specific 3′primer was designed from the cloned 3′end cDNA sequence, combined the 5′primer in the plasmid ofλTripEx2 of the cDNA library, with the template of cDNA library, the 5′end cDNA sequence was amplified by PCR. Removed the overlapping sequences of the two cDNA sequence, a novel antibacterial peptide gene cDNA sequence of Musca domestica was spliced,and analyzed the cDNA sequence with the help of the free bioinformatics software in NCBI.Results1.Constructed a antibacterial peptide gene chip and screened the antibacterial peptide associated genes of Musca domesticaPart insect antibacterial peptide gene sequences were acquired from the GenBank, according the design principles of oligo probe, with the help of the biology software Array Designer 2.0, 186 oligo hybrid probes were designed from the conservative sequences , the length were 50bp, melting temperature(Tm)were 72 to 77 degree,and the percent of GC were 40～60, which will be deposited on chips as a microarray in screening the antibacterial peptide associated genes of Musca domestica. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by E.coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analysised using the software of MIDAS,15 valid hybridization singals were detected through two times of hybridization and scanning, and the other 12 valid hybridization singals were also detected in the one hybridization and scanning, giving a clear positive control signal,whereas the negative and blank control were negative.2.Cloned and analyzed a novel antibacterial peptide gene of Musca domesticaA pair of degenerate primers were designed according the conservative domains in the amino acid orders of the insect antibacterial peptide gene Diptericin,which had been screened with gene chips in the first part, a 131bp homologous fragment was amplified by RT-PCR.according to the 5′and 3′primers in the plasmid ofλTripEx2 of the cDNA library of Musca domestic larve, a 366bp cDNA sequences of 3′end and a 457bp 5′end were amplified by PCR, at last, a 460bp novel antibacterial peptide gene cDNA sequence of Musca domestica was spliced from the 3′end and 5′end cDNA sequence,and this sequence were analyzed with the help of the bioinformatics software in NCBI,the result indicate that the protein encoded by this cDNA sequence belongs to Attacin gene family, the sequence of the 3′end is complete, existing a putative poly (A) tail signal and a poly (A) tail,but the 5′end is incomplete. Blast comparison showed higher gene homology exists comparing to Diptericin of Glossina morsitans morsitans and Protophormia terraenovae antibacterial peptide gene, 80% and 91%,respectively,the coded amino acid sequence comparison showed homology is 77% , 59% and 51%,comparing to Glossina morsitans morsitans, Drosophila melanogaster and Protophormia terraenovae. from the result of comparison, we can judge the cDNA sequence may be a novel antibacterial peptide gene of Musca domestica.Conclusion1. A antibacterial peptide gene chip of Musca domestica was constructed for the first time,and used this gene chip, twenty seven genes possibly related to antibacterial peptide from Musca domestica were screened initially, which established satisfactory foundation for furtherly cloning new antibacterial peptide genes of it.2. A 460bp antibacterial peptide gene of Musca domestica was successfully cloned, which had been screened with gene chips in the first part, and furtherly analyzed the cDNA sequence with the help of the free bioinformatics software in NCBI,which lay a solide foundation for the further research the Musca domestica antimicrobial peptide structure and functions, reveal their role in their innate immune system ,and then , prepare the Musca domestica antibacterial peptide through genetic engineering methods.
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