The Establishment Of The Prokaryotic Expression Method About Sheep ?-defensin-1(SBD-1)

Defensins are antibacterial peptides and they are important components of animal innate immunity.It has antifungal,bacterial and antiviral effects.It also has many biological functions such as killing tumor cells and so on.The related research has been in depth.But defensins have not been widely used because of difficulties in mass production.In this experiment,sheep defensins were expressed in vitro by two methods,and the antibacterial effects were studied to provide experimental basis for the application and industrialization of defensins.The results are as follows:Method 1: the fusion of expression methods with SUMOThe gene of Mongolia sheep beta defensin-1(SBD-1)was amplified by RT-PCR and connected to the SUMO gene.The recombinant plasmid was constructed and the recombinant plasmid was transferred into the expression of BL21(DE3)cells.By ultrasonic fragmentation,desalination,affinity chromatography,concentration,SUMO enzyme digestion,freeze drying and other techniques,the SBD-1 was obtained.Method two: expression method combined with elastin ELP(Elastin-like Protein,ELP).The full gene sequence of the mature peptide encoding SBD-1 was cloned by RT-PCR,and the gene was cloned into the prokaryotic expression vector pET-22 b,and the expression vector pET-22b-ELP-SBD-1 was constructed.The optimized sequence of the ELP-SBD-1 gene was obtained by optimizing the codon through the related software.On the basis of this sequence,the gene is synthesized.The optimized ELP-SBD-1 gene was transferred into the prokaryotic expression vector p ET-22 b,and the recombinant pET-22b-ELP-SBD-1 vector was obtained.The recombinant plasmid was transferred into the prokaryotic expression of BL21(DE3)E.coli,and the expression of ELP-SBD-1 fusion protein was induced by IPTG at different temperatures.SDS-PAGE analysis showed that fusion protein could be expressed at 37 ?.At 18 C,0.1 to 1 m M IPTG could induce fusion protein expression,and 0.5 mM IPTG induced more expression products.At 14-20 h time,the expression level of fusion protein increased with the prolongation of induction time,and the highest expression was induced when 16 h was induced.By means of ultrasonic fragmentation,ITC(inversetransition cycling,TC)of ELP(reversible phase transformation cycle)was purified to deter mine the optimum pH value and reversible phase transition temperature of the purified protein.The purified product was further purified after nickel column(desalting and affinity chromatography),and finally the SBD-1was successfully obtained through enrichment.The effects of SBD-1 on Escherichia coli and Staphylococcus aureus were verified respectively.Through bacteriostasis experiments,it was proved that the Bacteriostasis of Escherichia coli and Staphylococcus aureus could be formed,and the purity of SBD-1was obtained.