Expression And Preliminary Application Of Salmonella Pullorum Phage Lyase LySP2 In Pichia Pastoris

Salmonella pullorum is one of the most serious pathogens in the poultry industry,and it is also one of the 14 animal diseases that the Ministry of Agriculture requires to monitor.The purification of Pullorum disease infection is the best way to eradicate this disease.Antibiotics are traditional medicines for Pullorum disease.The seriously increased drug resistance has made the prevention and clearance of Pullorosis disease more difficult,due to the long-term abuse of antibiotics.Phage and its lyase have been widely studied and applied as novel antibacterial agents in improving food and medical safety.Therefore,screening and identification of high-efficiency phage and lyase of Salmonella pullorum are important for the prevention and purification of Pullrosis disease.A Salmonella phage YSP2 was isolated at the early stage.In this study,synthetic primers LySP2-ZF and LySP2-ZR were designed based on the sequence of the lyase LySP2 gene measured in the previous stage.The Xho I restriction site and the coding sequence AAAAGA were added at the 5'end,and the restriction endonuclease Xba I restriction site was inserted at the 3'end.After LySP2 and the vector pPICZ-?A were digested with Xho I and Xba I,the target fragment was ligated to construct the recombinant plasmid pPICZ-LySP2.The recombinant plasmid was linearized by Sca I digestion and electroporated into Pichia pastoris X33.High expression positive strain X33-pPICZ-LySP2 was obtained by PCR identification,sequence analysis and Zeocin⁺resistance screening.The fermentation conditions were optimized,and the optimum fermentation temperature of the recombinant yeast strain was 29°C.The fermentation supernatant was prepared at a concentration of 1.97 mg/mL by adding 1.5%methanol and inducing 96 h.The supernatant was analyzed by SDS-PAGE,and there was a band around 17 kDa.The percentage of the target protein LySP2 in the total protein of the supernatant was 12.12%,and the expression in the fermentation supernatant was238.76?g/mL.The lyase has high temperature resistance.The fermentation product was treated at 4-40°C for 30 min,and the enzyme activity was unchanged.At50-90°C,the enzyme activity showed a significant downward trend,and the remaining enzyme activity was about 54.17%.Meanwhile,the lyase can be adapted to a wide range of pH values,and the enzyme activity is maintained above 85%after treatment at pH 3-9.The activity assay showed that LySP2 had significant bactericidal activity under suitable conditions,and its thermal stability and acid-base resistance possess good application.Salmonella pullorum infection model of chicks was established by feeding 0.5mL of Salmonella solution?the concentration of Salmonella pullorum was 10⁶CFU/mL?to 3 day-old chicks.The results showed that continuous feeding of 10¹⁰PFU/mL phage or 60?g/mL lyase could achieve a survival rate of 70%in chicks,while the survival rate of supernatants with yeast control group or infection group was less than 30%.Therefore,the recombinant lytic enzyme LySP2 has certain clinical significance for the treatment of Pullorosis disease.The results showed that the liver of the chicks treated with lyase could completely eliminate Salmonella pulmonium and clear half of the Salmonella pulmonium in the intestine.Compared with the infection group,the clearance rate of Salmonlla in liver and intestinal was extremely different,indicating that the lytic enzyme can effectively remove Salmonella pulmonium in the chicks.In addition,the histopathological sections of the liver and intestine of the chick model were observed.The results showed that the recombinant lytic enzyme LySP2 can effectively alleviate pathological damage of organs.In conclusion,this study constructed the Pichia pastoris expression vector that efficiently expressed Salmonella pulmonium phage lyase and secreted the recombinant lytic enzyme LySP2.The recombinant lytic enzyme LySP2 has good antibacterial activity in vitro,and can significantly improve the survival rate of chicks,remove Salmonella pullorum in chicks,and reduce pathological damage of organs in vivo.LySP2 has great application potential in the treatment of Pullorosis disease by Salmonella pullorum,which lays a foundation for further evaluation on antimicrobial activity and application of the lyase.