| Defensins are small cationic peptides in antimicrobial peptides that are secreted in a variety of organisms,including mammals,fish,birds,insects,and plants.Defensins have a broad spectrum of antibacterial and antiviral activities.Defensins play an important role of innate immunity,they can also regulating the body’s immune system.Because of the relative deficiency of acquired immunity in fish,the study of fish defensins is very meaningful.According to the origin and structure of species,defensins can be divided into plant defensins,insect defensins and animal defensins.The study of β-defensin of Animals is the most profound and influence currently.In terms of fish,the study of defensins mainly focuses on the aspect of cloning and physicochemical properties.Genes of β-defensin have been cloned from grouper,barley,gingiva,loach,rainbow trout,tilapia and other fishes,and their expression in tissues has been turned out.Prokaryotic,eukaryotic expression and artificial synthesis were used to obtain the corresponding β-defensin mature peptide proteins,which have inhibitory effects on various bacteria.In this paper,we cloned the full-length cDNA sequence of grass carp β-defensin 1(DB1),grass carp β-defensin 2(DB2)and grass carp β-defensin 3(DB3).The serial numbers are KX906958.1,KX906958.1,KX906958.1 respectively.The full-length DB1 cDNA sequence is 705 bp and the open reading frame(ORF)is 204 bp.amino acid residues encoding 67.the first 24 amino acid residue is signal peptides.The isoelectric point of the mature peptide is 7.82,and the molecular weight is 4.5 KDa.The full-length DB2 cDNA sequence is 951 bp and the open reading frame(ORF)is 207 bp,amino acid residues encoding 68,the first 22 amino acid residues is signal peptides.The isoelectric point of the mature peptide is 8.65,the molecular weight is 5.3 KDa.The full-length DB3 cDNA sequence is 1110 bp.The reading frame(ORF)is 213 bp,amino acid residues encoding 70,the first 27 amino acid residues is signal peptides,the isoelectric point of the mature peptide is 8.89,and the molecular weight is 5.3 KDa.Using PCR amplification(specifically),we also cloned the full-length of grass carp DB1,DB2,and DB3,and found out that they all have three exons and two introns.The relative position of the signal peptides are the same as the mature peptides in the exons,and the arrangement of the 6 cysteines in the exons of grass carp are the same as other fishes.In order to research the distribution of β-defensin in the body of grass carp.we used RT-PCR to study the expression level of β-defensin.In healthy grass carp,we found that grass carp DB1 is highly expressed in eyes and skin;DB3 has a very high expression in eyes;DB2 expression in the eye is higher but significantly lower than DB1,DB3.The expression of other organs remained at a low level.To study the transcriptional regulation of grass carp β-defensins,we cloned the DB1,DB2,and DB3 partial promoter sequences,respectively.Software analysis revealed that there are multiple NF-κB regulatory sites in the DB1 promoter,no NF-κB regulatory site in the DB2 promoter,and a NF-κB regulatory site in the DB3 promoter.For this purpose,we constructed expression plasmids p65-ORF-pET32 a and its mutant proteins including N-terminus deletion mutant of p65-ΔN-pET32 a and C-terminus deletion mutant of p65-ΔC-pET32 a,and expressed prokaryotic expression p65(a major regulatory subunit of the NF-κB family)and p65 truncated proteins.In vitro gel retardation experiments showed that the purified recombinant protein p65 had a significant blocking effect on DB1 and DB3 promoter fragments.The cotransfection of pcDNA3.1-p65(or pcDNA3.1-p65-ΔC,pcDNA3.1-p65-ΔN respectively)with pGL-DB1-PF or pGL-DB3-PF and pRL-TK renilla luciferase plasmid into CO(Ctenopharyngodon Idella ovary)cells were found to significantly up-regulate pGL-DB1-PF luciferase activity.However,p65 down-regulated the luciferase activity of pGL-DB3-PF in the same experiment.In the study of antibacterial activity of grass carp β-defensins,we constructed DB1,DB2,and DB3 mature peptide sequences in prokaryotic expression vector pET32 a,respectively.After induced by IPTG,purified DB1,DB2,and DB3 recombinant proteins were obtained.DB1,DB2,DB3 mature peptide proteins were obtained by digestion with enterokinase.In the antibacterial experiment,DB1 and DB3 recombinant proteins were found to have no bactericidal activity,while DB2 recombination retained some of the protein activity and had a certain antibacterial activity against Bacillus subtilis. |
