Expression,Purification And Immunogenicity Evaluation Of REV-env Protein In Pichia Pastoris

Reticuloendotheliosis(RE)is a group of pathological syndromes caused by reticuloendotheliosis virus(REV).The main manifestations are growth retardation,acute reticulocyte tumors and chronic lymphoid tissues.REV is usually mixed with other viruses and easily integrated into the host genome.In addition,immunosuppression after infection has led to more and more difficulties in the prevention and control of RE.The aim of this study was to compare the immunogenicity of env protein and gp90 protein and to screen the proteins with better immunogenicity and lay the foundation for the study of the RE subunit vaccine.First,in this study,the envelope protein of REV was expressed by Pichia pastoris expression system.Recombinant strains containing different numbers of Menv expression cassettes were constructed by P.pastoris host strains SMD1168 and GS115,respectively,and induced to express in shake flasks.The results showed that the env protein can be successfully expressed in the constructed Pichia pastoris strain.The expression level in GS115 was significantly higher than that in SMD1168.The expression level of recombinant strain GS115/pAO815-2Menv was higher than that of GS115/pAO815-4Menv and GS115/pAO815-6Menv which shows that the increase in the number of expression cassettes cannot increase the expression of env protein.By optimizing the inducing conditions,we found that the recombinant strain GS115/pAO815-2Menv can express 0.5 g/L env protein in shake flasks.In order to obtain a large amount of recombinant env protein and gp90(supernatant)protein,the culture conditions of the two proteins were optimized,and the protein obtained from the fermentation was purified.Proteins before and after purification were stored at room temperature,4°C and-20°C,respectively,to compare their stability.The results showed that the env protein expression was the highest,reaching 3.5 g/L when the recombinant strain GS115/pAO815-2Menv was cultured at 29°C and pH=5.5.The recombinant strain SMD1168/pPIC9k-gp90 was fermented at 27°C and pH=6.0,and the gp90(supernatant)protein had the highest expression level up to 1.1 g/L.The high-purity env protein and gp90(sediment)protein can be obtained by affinity chromatography,and the gp90(supernatant)protein was concentrated by a 30 kD ultrafiltration tube.The stability of the three proteins were improved after purification,but it was not significant and there were still serious degradation problems.The immunogenicity of gp90(supernatant)protein,gp90(sediment)protein and env protein was compared by animal challenge protection experiments.The results showed that all three proteins produced higher levels of ELISA antibodies and neutralizing antibodies.Compared with the control group,the viral load of the gp90(supernatant)protein-immunized group was reduced by 2.36×10~5-fold,the viral load of the gp90(sediment)protein-immunized group was reduced by 6.61×10~3-fold,and the viral load of the env protein-immunized group was reduced by 6.08×10~3-fold.These instructions show that all three proteins have good immunogenicity.In this study,the expression of env protein in Pichia pastoris was achieved,we obtained a large number of recombinant proteins with good immunogenicity by optimizing fermentation conditions,which can effectively protect SPF chickens from infection by REV.