Total RNA was separated from the abdomen of house fly,after which had been challenged for 24 hours with E. coli and Staphylococcus aureus . Then,the mRNA was purified from it. After that,ds cDNA was synthesized by reverse transcriptase from mRNA,and small cDNA which was lower than 400?bp was removed by Sepharose CL 4B spun column. After Eoc RⅠ/ Not Ⅰ Adaptors were added to cDNA,those that weren’t ligated to cDNA were removed by another Sepharose CL 4B spun column. The cDNA was inserted into λgtll,the recombined vector packaged in vitro ,infected a host strain Y1090. Thus,the cDNA library was constructed. The titer of the newly constructed cDNA library was 3 46×10 5 pfu/mL,and its recombination rate was 99 6%. The library would provide basis for the cloning of the antimicrobial peptide genes of house fly.
Defensin is a kind of cationic, inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms. With their broad antimicrobial spectrum and strong pharmaceutical effects, antimicrobial peptides, including defensins, represent a source of novel antibiotic agents. A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDNA library of houseflies (Musca domestica) that had been challenged by E. coli and Staphylococcus aureus. Sequence analysis revealed that the open reading frame of the cDNA encoded a 92-amino acid peptide, which contained an NH 2-terminal signal sequence (1-22) followed by a propeptide and the mature peptide (53-92). The sequence identity with other insect defensin is between 51% and 73%. The mature peptide, with a predicted molecular weight of 4\^0 kDa, and pI of 8\^69, has 1 negative charged amino acid and 4 positice ones. The putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds, Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6. These results suggest that the novel full-length cDNA of the defensin gene, denominated Mdde, has been successfully cloned from houseflies.
