Expression And Activity Analysis Of Three Avian β-defensins In E.coli

In the poultry breeding industry,avian colibacillosis is a high-risk disease.The incidence is high in the case of irregular feeding management and poor breeding environment.The main symptoms are:air sacs,heart disease,fallopian tubes.Inflammation,etc.,caused a large economic loss to poultry farming,restricting the healthy development of the poultry industry,and Avain pathogenic Escherichia coli(APEC)is the main cause of avian colibacillosis.Avian beta defensins(AvBDs)are small peptides with a high content of cysteine.The molecular weight of the protein is usually around 3-6 kD,which plays an important role in the innate immune system of chickens.Due to its special antibacterial mechanism,it does not produce residues in animals,and has a broad spectrum of antibacterial properties,it is expected to become an antibiotic substitute.However,since defensins are expressed in a low amount in the body and the separation cost is high,an expression system is constructed in vitro to obtain a large amount of defensins.In this study,β-defensins:AvBD2,AvBD6 and AvBD9 were used as the research objects to synthesize these three defensin mature peptide genes,and the mature peptide gene sequences were optimized according to the codon preference of E.coli,and subcloned into The pET-32a vector was transfected into E.coli BL21(DE3)pLysS competent cells,and protein expression was induced by IPTG.The protein expression was analyzed by SDS-PAGE and examined by Western-blot.Protein purification was carried out with nickel column.Six APEC strains preserved in this laboratory were selected as indicator strains,and purified defensin protein was used for in vitro bacteriostatic test.The AE17 strain preserved in our laboratory was used for challenge protection test.Recombinant defensin protein activity,related research is as follows:1.Construction of three avian beta defensin vectors and protein expression and purificationThree avian β-defensin mature peptide gene sequences were obtained according to Genbank,optimized according to E.coli codon preference,subcloned into pET-32a expression vector,and then it was transformed into BL21(DE3)pLysS competent cells,using SDS Protein expression to analyzed by-PAGE,the target protein was purified using a nickel column and verified by Western-blot.Western-blot confirmed that AvBD2,AvBD6 and AvBD9 have specific bands at around 25 kD,which proved that the three fusion proteins were expressed in BL21(DE3)pLysS engineering bacteria.The concentrations of the three fusion proteins after purification were determined by the BCA protein concentration assay kit to be 0.491 mg/mL,1.14 mg/mL,and 1.025 mg/mL.2.In vitro antibacterial activity of three avian beta defensinsA sterile 96-well plate was selected,and each defensin was set to 2 replicates,and the OD600 absorbance value was measured every 2 h.The micro-antibacterial test results of AvBD2 showed that the growth of avian pathogenic Escherichia coli AE17 was inhibited by adding AvBD2.Sexuality,with the decrease of concentration,the inhibitory effect was also reduced,and the minimum inhibitory concentration was 150 mg/L.The results of micro-inhibition test of AvBD6 showed that the concentration of each protein had obvious effect on the growth of AE17 before adding it to AvBD6.Inhibitory,the minimum inhibitory concentration was 150 mg/L;the results of micro-inhibition test of AvBD9 showed that the overall effect was similar to that of AvBD6,and had a significant inhibitory effect on the growth of AE17 before 10 h,and the minimum inhibitory concentration was 300 mg/L.3.Three kinds of avian beta defensins in the treatment of chicks infected with APECThe 56 14-day-old chicks were randomly divided into 8 groups:AvBD2,AvBD6 and AvBD9 control group,AvBD2,AvBD6 and AvBD9 treatment group,blank control group and challenge group,with 7 rats in each group.Blank control group:1 mL sterile PBS was injected into each chick;challenge group:1 mL of intramuscular injection was given to each chicken according to the minimum lethal concentration of AE17;treatment group:1 mL of each chicken was intramuscularly injected according to the minimum lethal concentration of AE17 The solution was treated with recombinant AvBD2,AvBD6 and AvBD9 1 mL(concentration of 300 μg/mL)when the chicks developed clinical symptoms(about 6 hours later).After 7 days of observation under the same feeding conditions,chicks died in the blank control group;the mortality rate in the challenge group was 100%;the mortality rate in the AvBD2 treatment group was 14%;the mortality in the AvBD6 treatment group was 0%;and the death rate in the AvBD9 treatment group was 28%.The results indicated that recombinant AvBD2,AvBD6 and AvBD9 could better resist the infection of AE17 strain and reduce the mortality of chicks.