Prokaryotic Expression Of Chicken ?-Defensin Gal-4 Gene And Detection Of Antibacterial Activity Of Recombinant Protein

Defensins are developed along with people's understanding of the toxic and side effects of antibiotics.Many defensins have inhibitory effects on bacteria,fungi,viruses,etc.They are not easy to produce drug resistance and have good stability.They can be made into new antibacterial drugs or used as feed additives and food preservatives,etc.At present,the research on avian?-defensins is quite popular.Many chicken ?-defensins have been cloned and expressed,and usually have antibacterial activity.However,the research on cloning and expression of chicken?-defensins Gal-4 gene has not been reported.The author obtained recombinant protein of Gal-4by using genetic engineering technology,and identified its antibacterial activity.Finally,the prevention and treatment of bacterial infection in mice were studied by using this protein.It has made certain contributions to the development of new antibacterial drugs,provided experimental basis for screening and identifying defensins with good biological activity,and provided good experimental basis for the application of Gal-4 in veterinary clinic.The main research contents and results are as follows:Total RNA was extracted from chicken bone marrow,and cDNA was synthesized by reverse transcription.Specific primers were designed to amplify mature peptide region gene of chicken?-defensin Gal-4.The target gene was cloned to BamHI and HindIII double cleavage sites of prokaryotic expression vector pET-30 a.Prokaryotic expression vectorpET-30a-Gal-4 was constructed and transformed into competent cells of Escherichia coli BL21.After IPTG induced expression,bacterial liquid was detected by SDS-PAGE and Western blotting,and recombinant protein was purified by Ni column affinity chromatography and desalted and concentrated.The results showed that 148 bp target gene was successfully amplified and prokaryotic expression vectorpET-30a-Gal-4 was constructed.After induced expression,the target protein with14 kDa size could be detected.After purification,a recombinant protein with a concentration of0.8 mg/mL was obtained.SDS-PAGE showed a clear target band at 14 kDa.In order to identify the antibacterial activity of recombinant protein Gal-4 in vitro,a preliminary study was carried out using Staphylococcus aureus and Pasteurella multocida.The antibacterial activity of recombinant protein Gal-4 against Staphylococcus aureus and Pasteurella multocida was identified by perforation method and bacterial growth curve method.The results showed that recombinant protein Gal-4 had good antibacterial effect on Staphylococcus aureus and Pasteurella multocida.The recombinant protein Gal-4 was used as a new antibacterial drug to prevent and treat staphylococcus aureus infection in mice model.The results showed that the recombinant protein Gal-4 played a good bactericidal effect in mice and had a certain effect in preventing and treating animal diseases.