Cloning And Expression Of The MdDpt And MdAtt-2 From Musca Domestica

Antimicrobial peptides are a kind of small molecular peptides with antimicrobial activties, which play a critical role in the innate immunity system. Musca domestica can live in surroungings full of various microbes, because the houseflies have unique immune defense mechanism and can produce antimicrobial peptides with strong activity.This thesis study with housefly, contains 3 parts.Part 1: With the methods of RT-PCR and RACE techniques, 2 different genes cDNA sequence of Diptericin(MdDpt) and Attacin(MdAtt-2) were cloned from the housefly body, and its protein sequences were analyzed;Part 2: The house flyβ-actin gene as a internal control, using semi-quantitative RT-PCR to detect MdDpt and MdAtt-2 genes'mRNA expression level relative quantitative research of housefly after stimulation by the E. coli;Part 3: Construct the MdDpt and MdAtt-2 prokaryotic expression vector of housefly, and in E. coli (DE3) induced expression, and the purified protein DsbA-MdDpt was used to prepare New Zealand rabbit anti-serum.Results:(1) A full-length 410 bp gene cDNA sequence of the housefly MdDpt was cloned, which containing a 300 bp complete open reading frame (ORF), according to ORF deduced that MdDpt peptide has 99 amino acids, SignalP 3.0 program predicte N terminal signal peptide consists of 20 amino acids, MdDpt mature peptide contains 79 amino acids, theoretical molecular weight is 8.79 kD, theoretical isoelectric point is 8.27. The result of sequence alignments analysis showed that this gene shares 74% identity with Stomoxys calcitrans's Diptericin A.(2) The full-length of housefly MdAtt-2 gene is 809 bp, the open reading frame (ORF) is 726 bp, according to ORF deduced the MdAtt-2 polypeptide composed of 241 amino acids, SignalP 3.0 program predictes the N terminal includs a signal peptide of 22 amino acids, the mature peptide of MdAtt-2 has 219 amino acids, theoretical molecular weight is 23.63 kD, theoretical isoelectric point of 9.77. Homology analysis showed that this gene shares 45% identity with Sarcophaga peregrina's Sarcotoxin IIA.(3) Semi-quantitative RT-PCR analysis: data analysis showed that the housefly larvae without induced, the expression of two genes are very low volume, two gene expression increase rapidly in the 12 h peak after E. coli stimulation, and then gradually restored, 72 h after E. coli stimulation the 2 genes tended to normal levels by t test, two genes expression volume of 6-72 h after stimulation were higher than the control group, and have a significant difference (P <0.01).(4) Use the prokaryotic expression vector pET-DsbA, we construct the housefly MdDpt and MdAtt-2 prokaryotic expression vector successfully, and culture them in E. coli (DE3) at 37℃, the IPTG final concentration is 0.8mmol/L. SDS-PAGE protein electrophoresis reveal that the recombined protein of DsbA-MdDpt molecular weight is 33 kD, recombined protein DsbA-MdAtt-2 molecular weight is 48 kD, they are consistent with there theoretical molecular weight, they were purified by His affinity chromatography. MdDpt polyclonal anti-serum were prepared by immuning New Zealand rabbit with the recombined protein prepared, Western Blot showed that the anti-serum have good specificity.