Study of the feasibility of recombinant antimicrobial peptide expression and activity in vitro

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Wen, Lihua
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University of Guelph

Antimicrobial peptides (AMPs) are small peptides that are capable of inhibiting a broad spectrum of microorganisms. AMPs may have significant applications for human disease prevention and animal production as a substitution for antibiotics. The current study demonstrated the expression of bovine tracheal antimicrobial peptide (bTAP) and cecropin P1 (CP1) in porcine tracheal epithelial cells and Cos7 cells. The expressed CP1 is secreted into culture medium, and has activity against 'E. coli in vitro.' DNA sequence encoding the mature form of CP1 was synthesized and cloned into the vector pSGHVO, which contains the human growth hormone (hGH) gene sequence driven by the SRa promoter. The expression vector was introduced into both pTEC and Cos7 cells via Lipofectamine transfection. RT-PCR revealed that CP1 mRNA was present in both pTEC and Cos7 transfected cells. To study if the recombinant protein was translated and secreted, western blots were performed against His-tag. The 27 kD bands were detectable in both of the cell lysate and culture medium of transfected Cos7 but not pTEC. To further study if the secreted CP1 has antimicrobial activity, CP1 was released from the fusion protein by tobacco etch virus (TEV) protease, and radial diffusion assay (RDA) was performed. Antimicrobial activity against 'E. coli ' was detected in medium of pSGHVO-CPI transfected Cos7 cells. In conclusion, bTAP and CP1 can be transcribed, translated and secreted into culture medium in mammalian epithelial cells, and the secreted CP1 has antibacterial activity 'in vitro.' Our study demonstrated the feasibility of producing nematode-derived antimicrobial peptides in mammalian cells. The results are useful for further study to generate disease resistant transgenic animals using CP1 as transgene.

antimicrobial peptides, expression, bovine tracheal antimicrobial peptide, cecropin P1, porcine tracheal epithelial cells, Cos7 cells