Cloning And Expression Pattern Of Antibacterial Peptide Diptericin From Musca Domestica

The unique immune system of most living organisms was formed to combat microbial challenge in Long evolutionary process.In higher vertebrates,the host defense system depend on two systems: the innate immunity and adaptive immunity. But immunity of the insect that widly distributed in nature only depend on innate immunity,such as antimicrobial peptides-widely in eukaryotes and prokaryotes in vivo and molecular weight of the small, high isoelectric point, good thermal stability, a broad spectrum of the cationic peptide. They not only appeared to kill the G+ bacteria and G- bacteria, but also played a role of anti-fungal ,anti-parasite, anti-tumor and anti-drug-resistant strains. At present, the cause of the abuse of antibiotics and some have already declared many years ago with the extinction of microbial resistance and resurgence, with the new emerging microorganisms, the development of new antibiotics of the variation can not keep up the pace of pathogenic microorganisms, and the emergence of antimicrobial peptides undoubtedly troubled anti-infective treatment sheds some light, and insects in the agricultural research on the antimicrobial peptide, can better control pests. Therefore, the study of antimicrobial peptides have a very important value.Musca domestica are Diptera (diptera) Muscidae (muscidae) insects, in addition to harassment, and contamination of food, but also a media of variety of important human diseases,there is the spread of virus, bacteria, protozoa and so on, have done great harm to humanity. Housefly to carry many pathogenic microorganisms and themself were not infected by the disease has aroused great concern. The pre-lab has cloned a cecropin gene (Genbank accession number: EF175878), a defensin gene (Genbank accession number: EF175879), a attacin (Genbank accession number: DQ062744) gene, have a gene chip technology to screen the diptericin fragment.In this study, RACE technology, based on the laboratory prior to screening by gene chip technology to diptericin from housefly antimicrobial peptide gene fragments, cloning a four amino acid differences in both the full-length genes and diptericin its separately related to the study of biological information analysis; The use of real-time fluorescence quantitative PCR (Real-time PCR) and in situ hybridization was made in time and space of their expression patterns, compared before and after induction, different time, different stages of growth and development capacity diptericin gene expression changes in the study, and three in the house fly larvae in vivo expression of the organizations studied; used prokaryotic expression system pGEX-4T-1, respectively, made their prokaryotic expression, and Westernblot made identification of the expression products.The results of this study are as follows:1.Using different RACE methods, respectively, using gene-specific primers and universal primers from the success of immune-induced 3rd-instars larvae of the three has been both a total length of 433bp and 468bp (Genbank accession number, respectively: FJ748596, FJ795370) gene of diptericin cDNA, which has two full-length cDNA encoding a complete frame, encoding 99 amino acids. Bioinformatic analysis related results show that with the stable fly sting gene homology diptericinA highest 77%. And separately for its detailed study of the biological information analysis.2.Based on cloned full-length gene diptericinâ… , a pair of primer were designed of the uninfected, infected after 2h, 4h, 8h, 12h, 24h, 36h, 48h of housefly 3rd-instars larvae for real-time PCR, test results show that: expression of diptericin of the uninfected larvae was very low, 2h after induction ,it began to increase , reached the peak after 12h, the slow decline back to 48 hours after 24h compared to 2h; the same primers used for the non-induced larvae of the 3instar housefly different stages of development of real-time PCR testing, results showed that:The lowest level of transcript detected for all stages diptericin gene was in the eggs,1st-,2nd-instar and pupae,and the greatest level of mRNA for all stages was in the 3rd-instars.3.In order to study the expression pattern of different tissues of the diptericin from Musca domestica larvae that were infected the mixtures of multi-drug resistant bacteria,in situ hybridization with diptericin gene anti-sense DIG RNA probes was performed to detected the transcript level. The results showed that there was strong transcription in the fat body and that transcript was also detected in the midgut,traehea and Malpighian tubes. However,transcript was not detected in musceles,body wall,in uninfected flies,there were weak expression in traehea and Malpighian tubes.4.Based on the cloning of two full-length gene diptericin gene, construct prokaryotic expression vector to obtain fusion of glutathione (GST) tag of the recombinant plasmid pGEX-4T-1/diptericin, transformed into E. coli host cells induced by expression during the induction of IPTG. Using SDS-PAGE and Western blotting to identify the expression of the target protein .