Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri.
Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking. The full-length DNA of the scallop H2A was 696bp long, including a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 228bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated.
Li
Chenghua
C
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China.
Song
Linsheng
L
Zhao
Jianmin
J
Zhu
Ling
L
Zou
Huibin
H
Zhang
Huan
H
Wang
Hao
H
Cai
Zhonghua
Z
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D001483:Base Sequence; D003001:Cloning, Molecular; D006434:Hemocytes / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016982:Micrococcus luteus / Q000187:drug effects; D049897:Pectinidae / Q000235:genetics* / Q000378:metabolism* / Q000382:microbiology; D010843:Pichia / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000633:toxicity; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014733:Vibrio / Q000187:drug effects
D000900:Anti-Bacterial Agents; D006657:Histones; D012333:RNA, Messenger; D011994:Recombinant Proteins