Transient Expression of Chicken Antimicrobial Peptides by Mouse Mammary Carcinoma Cells C127
Abstract:Fowlicidin-3 and fowlicidin-1 are cathelicidin-type antimicrobial peptides found in chicken. They effectively inhibit the proliferation of many gram-positive and gram-negative bacteria. To obtain sufficient amounts of these peptides for possible use in therapeutic applications, DNA encoding each full-length gene, including all exons and introns, was fused to the β-casein promoter in a pBC1 vector that was then introduced into C127 cells. The full-length precursor proteins were expressed in response to a mixture of insulin, hydrocortisone, and prolactin. Processed fowlicidin-1 and fowlicidin- 3, as well as their precursors, were found in the cell culture media, which suggested that they could be processed and secreted. These transgenic peptides had antibacterial activity. Thus, transfected C127 cells may serve as an in vitro transgenic cell system that can be used to evaluate if specific gene constructs can be efficiently expressed in the mammary glands of transgenic mice.
Keywords: Anti-rabbit immunoglobulin G; Antibacterial activity; Antimicrobial peptides; BCA Protein Assay Reagent kit; C127 cells; DNA sequencing; EDTA; Escherichia coli; Fetal bovine serum; Fowlicidin-3; Human β-defensin 2; Mouse Mammary Carcinoma Cells C127; Na3VO4; PMD19T vectors; Reverse Transcription Real Time-PCR Analysis; Staphylococcus. aureus; Triton X-100; UV- spectrometry; XhoI-cleaved pBC1; ampicillin; antimicrobial peptide; cDNA; cathelicidin-type antimicrobial peptides; cell lysis buffer; eukaryotic expression systems; fowlicidin-1; fowlicidin-3; gel electrophoresis; glyceraldehyde 3-phosphate dehydrogenase; gram-negative; gram-positive; horseradish; human cathelicidin LL-37; hydrocortisone; immunoprecipitation; insulin; leupeptin; pBC1; pBC1 vector; pBCFow-1; pBCFow-3; polymerase chain reaction; prolactin; real time (RT)-PCR; sodium dodecyl sulfate; sodium pyrophosphate; transgenic mice; transient expression; trichloroacetic acid method; vector; western blotting; β-casein promoter; β-glycerophosphate
Document Type: Research Article
Publication date: December 1, 2010
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