The Four Freshwater Fish Histone H2B And Its Derived Antimicrobial Peptide HLP-1Gene Cloning, Characteristic Analyses And Prokaryotic Expression

With the increase of drug tolerance of bacteria, specially, for antibiotics, has been paid more attentionsin practice. Therefore, screening the new effective antimicrobial drug, taking the place of the traditionalantimicrobial drug, become the focus of current study in aquaculture. Antimicrobial peptides play animportant role in improving fish antibacterial ability and prevent fish from incurring disease. Histone H2Band its derived antibacterial peptide HLPs, as one part of natural immune, have important antimicrobialability. This study aims to analyze the characteristics of histone H2B and its derivatives antimicrobialpeptides HLP-1, and and to further clarify antibacterial mechanisms at the molecular level.In this study, according to the conserved sequences of zebrafish (Danio rerio) and other related fishH2B genes, primers were designed and optimized. Histone gene H2B was amplified through PCR methodin the bighead carp (Aristichthys nobilis), grass carp (Ctenopharyngodon idellus), common carp (Cyprinuscarpio), crucian carp (Carassius auratus), respectively. The approximately length375bp of histone H2Bgene sequence was obtained by sequencing, repectively. Similarity comparisons among nucleic acidsequences were implemented using the software DNAMAN. The results showed that the homology wasabove90%. Online analyses of the encoded protein H2B showed molecular weight was10KDa to15KDa.Secondary structure prediction showed that histone H2B was composed of random coil and α helixstructure. Tertiary structure prediction showed that α-helix composition in histone H2B was more than50%.In this study, the target gene H2B and expression vector PET-32a (+),were digested by the doublerestriction endonucleases, respectively, then connected to form expression vector PET-32a-H2B, whichwas transformed into E. coli BL-21(DE3), Protein H2B was induced to express by IPTG stimulation. Theresults of SDS-PAGE electrophoresis indicated that protein H2Bs of crucian carp and common carp weresuccessfully expressed in prokaryotic cells BL-21(DE3)Based on the gene H2B of Bighead carp, grass carp, common carp and crucian carp, primers weredesigned and optimized to amplify H2B derived antimicrobial peptides HLP-1gene.The character ofdeduced protein sequence of H2B derived antimicrobial peptides HLP-1was analyzed through online websites and related software. The results showed that HLP-1was about2KDa of molecular weight, andcontained a lot of cations with positive electric charge. Secondary structure of HLP-1consisted of randomcoil and α-helix structure. Tertiary structure of HLP-1showed that HLP-1was situated in the aminoterminus of the histone H2B.The target gene HLP-1and vector PET-32a(+) were digested by double restrictionendocleases,respectively, then connected to form expression vector PET-32a-HLP-1, which wastransformed into E. coli BL-21(DE3).Protein HLP-1was induced to express by IPTG stimulation. Theresults of SDS-PAGE electrophoresis indicated that protein HLP-1of grass carp was successfullyexpressed in prokaryotic cells BL-21(DE3).This study has successfully cloned histone H2B gene and its derived antimicrobial peptide HLP-1gene, as supplied the foundation for further study on H2B and its derived antimicrobial peptide HLP-1infish. Based on the deduced protein sequence of H2B and HLP-1, protein structure and characteristic wereanalyzed onlinely to supply the basis for exploxing the origin and mechanism of antimicrobial HLP-1.Histone H2B of crucian carp and common carp, and HLP-1of grass carp were successfully expressed inprokaryotic expression plasmid by IPTG stimulation in E. coli BL-21(DE3), the target proteins wereseparated by SDS-PAGE electrophorosis, which can provide a system for screening the higher effectiveantimicrobial peptides.