An Efficient Protocol for DNA Amplification of Multiple Amphibian Skin Antimicrobial Peptide cDNAs
Antimicrobial peptides (AMPs) play an important role in the host’s innate defence system in many organisms. Amphibian skin is expected to be a particularly rich source of novel AMPs. In amphibians, AMPs are produced from precursor proteins via specific cleavage by processing enzymes. While the nucleotide sequences of the AMP coding region in precursors are hypervariable, those of other regions, including the 5′- and 3′-untranslated regions (UTRs), are highly or relatively conserved in different precursors. Such nucleotide sequence conservation suggests an efficient strategy for molecular cloning of the antimicrobial peptide genes by 3′-rapid amplification of cDNA ends (3′-RACE) and reverse transcriptase polymerase chain reaction (RT-PCR) methods using specific primers. With this strategy in mind we have established an efficient protocol suitable for amplification of multiple cDNAs encoding amphibian AMP precursor proteins.
Key wordsAmphibian skin antimicrobial peptides molecular cloning 3′-RACE RT-PCR
The authors wish to thank the members in the laboratory of Regulatory Biology in Toho University for their useful suggestions for the preparation of the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research (19570063) from the Japan Society for the Promotion of Science to S.I. and T.K.
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