Preliminary Study On The Antibacterial Activity And Mechanism Of Ctenopharyngodon Idella β-defensin 1 Against Three Pathogens

Vibrio mimicus,Aeromonas hydrophila and pathogenic Escherichia coli are common and important pathogens in aquaculture.In recent years,the frequent occurrence of fish bacterial diseases caused by these pathogens have become one of the bottlenecks restricting the healthy development of fish aquaculture in China.Due to the long-term and high-dose use of antibiotics in aquaculture,bacteria develop drug resistance have drug resistance,which results in little curative effect.Therefore,it is urgent to develop safe and efficient antibiotic alternatives.β-defensin1 is an antimicrobial peptide that exists in a variety of fish and has broad-spectrum antibacterial and immunoregulatory effects.Compared with traditional antibiotics,β-defensin1 is considered to be the most promising antibiotic substitute because of its unique antibacterial mechanism and difficult to make bacteria resistant.However,the antibacterial mechanism of the antimicrobial peptide against specific pathogens is still unclear,which seriously hinders its application in the prevention and treatment of bacterial diseases.Therefore,Ctenopharyngodon idellaβ-defensin1（Ciβ-defensin1）was served as the research object in this topic to study its antibacterial activity and mechanism against the above three pathogens with the help of related technologies such as bacteriology,cell and molecular biology,aiming to provide new methods for the prevention and treatment of fish bacterial diseases.The main research contents and results are summarized as follows.1.Recombinant expression of Ciβ-defensin1 proteinIn this paper,a pair of specific primers with restriction sites were designed according to the sequence of Ciβ-defensin1 gene and p ET-32a（+）vector.Ciβ-defensin1 gene was amplified by PCR using the pc DNA3.1-Ciβ-defensin1 preserved in our laboratory as the template.Then,Ciβ-defensin1 gene was cloned into the prokaryotic expression vector p ET-32a（+）to construct the recombinant prokaryotic expression plasmid p ET-32a（+）-Ciβ-defensin1 by enzyme-linked enzyme method.The recombinant plasmid was identified by PCR and double enzyme digestion,and a target gene DNA band with a size of about 153 bp was obtained.The sequencing results also showed that there was no mutation or deletion ofβ-defensin1 gene,indicating that the above recombinant prokaryotic expression plasmid was successfully constructed.Finally,the recombinant strain Rosetta-gami2（DE3）plys S/p ET-32a（+）-Ciβ-defensin1 was induced by IPTG and analyzed by SDS-PAGE.The soluble expression product was purified by using Ni-NTA agarose affinity chromatography column.After dialysis,concentration and protein concentration determination,the r Ciβ-defensin1 protein with purity of about 95%and concentration of 0.81 mg/m L was obtained.2.Detection of the antimicrobial activity of r Ciβ-defensin1 protein against three pathogensIn this paper,V.mimicus strain 04-14,A.hydrophila strain BA1 and pathogenic E.coli strain YE-1 preserved in our laboratory were first recovered,and their morphology and staining characteristics,culture characteristics as well as antigenicity were identified,respectively.Then,the antibacterial activity of r Ciβ-defensin1 protein against the above three bacteria was detected by drilling method,microdilution method and coating counting method,respectively.In addition,whether the recombinant protein was toxic to CIK cells was detected by CCK8 method.The results showed that the inhibition zone diameters of r Ciβ-defensin1 against three pathogens were 24±0.15 mm（V.mimicus）,22±0.20 mm（A.hydrophila）and 16±0.17 mm（pathogenic E.coli）,respectively.MIC values were 19.63μg/m L（V.mimicus）,39.25μg/m L（A.hydrophila）and 78.5μg/m L（pathogenic E.coli）,respectively;MBC values were 157.04μg/m L（V.mimicus）,341μg/m L（A.hydrophila）and>628μg/m L（pathogenic E.coli）,respectively.The survival rate of CIK cells treated with r Ciβ-defensin1 protein at the concentration less than or equal to 175μg/m L reached more than 96%.The results suggested that the antimicrobial peptide Ciβ-defensin1 had antibacterial activity against V.mimicus,A.hydrophila and pathogenic E.coli in vitro,and the antibacterial activity against V.mimicus>A.hydrophila>pathogenic E.coli,and had little cytotoxicity to CIK cells.3.Preliminary study on the antibacterial mechanism of Ciβ-defensin1 against three pathogensIn this paper,in order to preliminarily explore the antibacterial mechanism,the effects of r Ciβ-defensin1 protein on cell wall integrity,cell membrane permeability,intracellular DNA and protein synthesis of the three pathogens were detected by the OD₆₃₀value determination method,ONPG test,gel block test and BCA method,respectively.The results showed that r Ciβ-defensin1 protein could damage the cell wall and increase cell membrane permeability of the three pathogens.It could bind with genomic DNA of V.mimicus and pathogenic E.coli,but not A.hydrophila.It could inhibit the intracellular protein syntheses of the three pathogens.The results indicated that the antimicrobial peptide Ciβ-defensin1 played an anti-V.mimicus and E.coli role by enhancing the permeability of their cell wall and cytomembrane,and interfering with the syntheses of intracellular DNA and protein.While the antibacterial effect on A.hydrophila mainly involved in increasing the permeability of cell wall and cytomembrane,and inhibiting intracellular protein synthesis.In summary,on the basis of successful expression of r Ciβ-defensin1 protein,it was clarified that antimicrobial peptide Ciβ-defensin1 had antibacterial activity against the above three pathogens in vitro,and the mechanism of the antibacterial peptide against three pathogens was preliminarily discussed in the study.The results can provide new methods for the prevention and treatment of fish bacterial diseases,and will also promote the development and application of fish-derived antimicrobial peptideβ-defensin1.