Separation, identification and gene expression analysis of PmAMP-1 from Pinctada fucata martensii

Antibacterial peptides (AMPs) constitute an important part of the body's innate immune system and are responsible for a wide range of inhibitory effects against pathogens such as bacteria, fungi, and viruses. In this study, multi-step high performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify proteins with antibacterial activity from the serum of Pinctada fucata martensii (P.f. Martensii) and obtain a component named P.f. Martensii antimicrobial peptide-1 (PmAMP-1). PmAMP-1 cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) and mRNA expression of was analyzed by quantitative real-time PCR (qRT-PCR). From the results of this study, full-length PmAMP-1 cDNA was shown to be 700 base pairs (bp) long with an open reading frame (ORF) of 294 bp, encoding 97 amino acids with a predicted structure that is mostly α-helices. PmAMP-1 mRNA was constitutively expressed in all tested tissues including the adductor muscle, mantle, hepatopancreas, gill, gonads and hemocytes. The highest level of PmAMP-1 transcription was observed at 8 h and 2 h after bacterial challenge in hemocytes and adductor muscle (p < 0.01), respectively. Furthermore, PmAMP-1 caused significant morphological alterations in E. coli, as shown by transmission electron microscopy (TEM). The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.