Transient Expression of Chicken Antimicrobial Peptides by Mouse Mammary Carcinoma Cells C127

Na      Yi; Ning      Li

doi:10.2174/0929866511009011517

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: Antibacterial activity, antimicrobial peptide, fowlicidin-1, fowlicidin-3, pBC1, vector, transient expression, Mouse Mammary Carcinoma Cells C127, cathelicidin-type antimicrobial peptides, gram-positive, gram, –, negative, β-casein promoter, pBC1 vector, insulin, hydrocortisone, C127 cells, prolactin, transgenic mice, Antimicrobial peptides, eukaryotic expression systems,, Human β-defensin 2, human cathelicidin LL-37, Fetal bovine serum, PMD19T vectors, BCA Protein Assay Reagent kit, cell lysis buffer, western blotting, immunoprecipitation, Anti-rabbit immunoglobulin G, horseradish, polymerase chain reaction, Escherichia coli, XhoI-cleaved pBC1, pBCFow-1, pBCFow-3, gel electrophoresis, UV- spectrometry, Reverse Transcription Real Time-PCR Analysis, cDNA, real time (RT)-PCR, glyceraldehyde 3-phosphate dehydrogenase, trichloroacetic acid method, Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin, sodium dodecyl sulfate, Staphylococcus. aureus, ampicillin, DNA sequencing