CDNA Cloning And Sequence Analysis Of Antibacterial Peptide Of BTI-Tn-5B1 Cell Line

Antimicrobial peptides is a kind of small MW polypeptide which have the antimicrobial activity. They are encodeed by some special genes and exist in the innate immunity systerm of many organism. They widely exist in insect,amphibian, crustacean,mollusk,pisces,bieds,plants,mammal and so on . These peptides had the wide range of antibacterial,antiviral spectrum and inhibiting growth of tumor cell.The first antimicrobial peptides was isolated from insect. Various insects are known to produce humoral antibacterial proteins in response to injection of dead or live bacteria. Inscet antibacterial peptides were bioactive peptides,which played an important role in insect immunity. They are the first defense against environment infection for the organism.The propagation of immune-responsive cells in vitro has provided the basis for substantial contributions to our understanding of many aspects of the immune response. In contrast, the potential for exploring the innate immune response of insects using cultured cells is only beginning to be developed. Many experiments have proved that many insect cell lines in vitro(Drosophila melanogaster, Sacrophage pergrime, Anthonomus grandis, Aedes albopictus, Trichoplusia ni) were induced by treatment with lipopolysaccharide, lamimarin, flagellin and heat-killed Escherichai coli can produce antimicrobial peptides.So, we induced antibacterial peptides from cultured cells.In previous work, three cultured cells from Lepidoptera were stimulated with heat-killed Escherichia coli, antibacterial assays showed that the antibacterial activity of induced BTI-Tn-5B1 cell line (5B1) was highest, the antibacterial peptide with a molecular weight of 7490KD, pI 4.17, induced from BTI-Tn-5B1 cell line was a new kind antibacterial peptide.In order to study the cDNA structure of this new peptide,we detected the antibactrial activity of the 5B1 cell secretion and then used Tricine SDS-PAGE electrophoresis to separate the new peptide.After transmembrane in PVDF,sequencing its amino acids sequenece of N-terminatio by Edman degradation. Then we design a 3' RACE GSP ,and extract the total RNA from the 5B1 cell.We obtained the cDNA sequence by 3'RACE.and then analysed it in bioinformatics. Our results are as following:(1) The result of activity assay of induced supernant from 5B1 cell showed that the induced production from its secretion supernatant can have activities against microbe.(2) Tricine SDS-PAGE found that a new small peptide, with a molecular weight of about 8000Da, was preferentially induced in 5B1 cell.(3) Its amino acids sequenece of N-terminatio is A-V-N-V-E-T(A)-I(D)-A-A-V.(4) The cDNA gene of the new peptide was successfully isolated from 5B1.it contains 430bp bases.this gene encodes a peptide of 72 amino acids with 7920kD and contains a 216bpopen reading frame(ORF) and 214bpçš„3'UTR.(5) Bioinformatics analysis indicated that it has a high homology with ribosome gene.