Optimization Of Expression Conditions And Fermentation Process Of G2 Protein Of Micropterus Salmoides Rhabdovirus

Micropterus salmoides rhabdovirus（MSRV）disease is a kind of viral disease seriously harmful to the healthy culture of Micropterus salmoides.Vaccination as a green,safe and efficient means of prevention,it has achieved good results in the prevention and control of aquatic viral diseases,and has been widely paid attention.In this study we has a good immune effect of MSRV G2 protein vaccine,on the basis of G2 gene codon optimization,efficient prokaryotic expression vector filter expression,and conditions optimization of fermentation process optimization and so on research,aimed at improving G2 proteins the prokaryotic expression of quantity and scale of fermentation production,provides reference for building high yield and low consumption production line,Reduce the cost of vaccine production in the field.1.Codon optimization and expression vector selection of Micropterus salmoides rhabdovirus G2 proteinCodon optimization was carried out on the basis of G2 protein with good immune effect determined by our laboratory in the early stage,and the optimized sequence was compared with the original sequence by CHIPS and CUSP programs in EMBOSS software and Codon W1.4.2 software.Codon Adaptation Index（CAI）,Effective Number of Codon（ENC）and GC3s values were used to determine the appropriate expression host,and the results showed that:The CAI value of the optimized sequence increased from 0.54 to 0.78,the GC_3svalue increased from 45%to 55%,and the ENC value decreased from 47.99%to 44.77%.Moreover,only 5 of the optimized sequences differed greatly from the use frequency of the E.coli codon,indicating that E.coli was the most suitable expression host.The optimized sequence G2-opti was ligated with five candidate expression plasmids p ET-25b、p ET-28a、p ET-32a、p GEX-6P-1、p Cold I,ensuring that other conditions were the same,to determine the optimal expression vector.The results showed that using p ET-32a as prokaryotic expression vector was more conducive to the expression of exogenous genes in E.coli.2.Optimization of G2 protein expression conditions and verification of immune effectIn order to further improve the expression yield of G2 protein,single factor test was used to determine the optimal conditions of three factors affecting the protein expression（concentration of inducer,induction temperature,induction time）.On this basis,box-Behnken central combination test was used to explore the interaction among the three factors.Response Surface Methodology（RSM）was used to determine the comprehensive effects of the three factors on the yield of the target protein,and Design Expert 8.0.6 software was used to screen the optimal expression conditions for G2 protein prokaryotic expression.Response surface methodology was used to optimize the expression conditions.Under laboratory conditions,when the concentration of inducer was 0.8 mmol/L,the induction temperature was 29℃and the induction time was 5 h,the yield of target protein was increased from 40.5%to 60.1%,and the protein concentration was increased from 0.53g/L to 0.75g/L.The codon optimized G2protein was used for immersion immunization,and different immune concentrations（5mg/L,10mg/L,40mg/L）and PBS were set for control.Serum was collected and antibody titer was determined.The results showed that the G2 protein optimized by codon still had immune protection effect after immersion immunization.3.Optimization of recombinant G2 protein fermentation process of E.coliIn order to obtain sufficient amount of target protein in industrial production,this study carried out small-scale G2 protein fermentation in a 5L fermentation tank,and studied the effects of inoculation amount,culture temperature,supplementary medium formula,induction temperature and concentration of inducer on the yield of target protein,laying a foundation for subsequent large-scale production.The results showed that when the inoculation amount was 5%,the culture temperature was 40℃,the supplementary medium formula was 60 g tryptone,30 g yeast extract,60 g sodium chloride,60 g glucose,the concentration of inducer was 0.8 mmol/L,the induction temperature was 30℃,the maximum wet weight of the bacteria was 58.9g/L,the protein concentration was 2.03g/L.It provides an important reference for the preparation of G2 protein by large-scale fermentation in the later industry.In conclusion,this study improved the yield of G2 protein through codon optimization,expression vector selection,protein expression conditions and fermentation process optimization,which provided reference for large-scale production of vaccines and was of great significance for the promotion and application of fishery vaccines.