[Objective]To clone a novel antibacterial peptide gene from Penaeus monodon(PmALF-like),and to clarify its structural characteristics,tissue expression pattern,and its expression changes after stimulation of Vibrio parahe-molyticus,so as to explore its role and function in antibacterial innate immunity,with an aim to provide reference for eluci-dating the disease-resistant immune mechanism of P.monodon.[Method]Based on the expression sequence target(EST)sequence of PmALF-like gene,the full-length cDNA sequence of PmALF-like was obtained by RACE amplification.Bio-informatics analysis was performed using online softwares such as ProtParam,SignalP 5.0,MegAlign and MEGA 7.0.The expression characteristics of PmALF-like gene in different tissues and its expression changes after V.parahemolyticus stimulation were detected by real-time fluorescence quantitative PCR(qRT-PCR).The recombinant protein PmALF-like or mature peptide was obtained by prokaryotic recombination method(removing signaling peptide),and the inhibitory ef-fect of PmALF-like mature peptide on bacterial growth was investigated by liquid medium method and Oxford cup method.[Result]The total length of the cDNA sequence of PmALF-like gene was 1242 bp,including the 5'non-coding re-gion(5'-UTR)of 45 bp,the 3'non-coding region(3'-UTR)of 798 bp and the open reading frame(ORF)of 399 bp,en-coding 132 amino acids.The molecular weight was 15.26 kD and the theoretical isoelectric point(pI)was 7.67.A signal peptide of PmALF-like protein was predicted by the online software SignalP 5.0 with a splicing site at the 18th amino acid.The results of qRT-PCR showed that PmALF-like was expressed in hemopancreas,hepatopancreas,gills,intestinal tract,muscle and other tissues to varying degrees,with the highest relative expression level in hepatopancreas,followed by he-mopancreas,and less relative expression level in muscle tissue.After stimulation by V.parahemolyticus,the expression level of PmALF-like gene in hepatopancreas of P.monodon produced immune response,and its gene expression was sig-nificantly(P<0.05)or extremely significantly(P<0.01)up-regulated at 6-72 h.PmALF-like mature peptide without signal peptide was successfully obtained by prokaryotic expression method in vitro.It was found that the mature peptide of PmALF-like had obvious inhibitory effect on the growth of Aeromonas victori,V.parahemolyticus and Aeromonas hy-drophila by liquid medium method and Oxford cup method.[Conclusion]PmALF-like gene is widely expressed in various tissues of P.maculatus,its mature peptide has obvious inhibitory effects on the growth of A.vickeri,V.parahaemolyticus and A.hydrophila.
维普
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