Optimization Of Prokaryotic Expression Conditions And High-cell-density Cultivation Of Leptospira Interrogans Trigeminy Genus-specific Protein Antigen

Background and Objective:Leptospire belongs to Leptospiraceae of Leptospira,and is prokaryotic organisms.Leptospirosis,caused by infection of Leptospira interrogans,is a widespread zoonosis in the world.These highly invasive spirochetal pathogens are capable of infecting a broad range of mammalian host,murine,frog and pig are the most dangerous mammalian host to the people now.L.interrogans could live in water for weeks,people often infected through either direct contact with an infected animal or indirect contact with soil or water contaminated with urine from a chronically infected animal.Leptospirosis is the most widespread and dangerous zoonoses in the world.Inoculating vaccine is the most effective measure to prevent and control Leptospirosis.But the serogroups of L.interrogans are numerous,and there is a low or even no cross-protection among them.Therefore the epidemically dominant serogroups of L.interrogans have been selected for preparing polyvalent vaccine production.However,this kind of vaccine offers little protection against other serogroups of microbe that were not selected in the vaccine and there were serious side effects for this kind of vaccine too.So it is very important to find out the genotype antigen of L.interrogans to prevent and control Leptospirosis. Research has shown that the OMPL 1/2,lipL21 and lipL32/1 genotype include the major epidemic L.interrogans serogroups of China.All the recombinant expression protein could produce high titer of antibodies though immune rabbits,each of them can be cross-agglutination of all strains and block L.interrogans to adhere host cell.Therefore we think that OmpL1/2,LipL21 and LipL32/1 are specific genotype surface protein antigens with good antigenicity and immunoreactivity.In order to improve the immunogenicity of antigen,reduce the number of fermentation and lower production costs,we have constructed the rOMPL1/2-rLipL21 and rLipL32/1 fusion gene expression system of E. coli.E.coli is commonly used recombinant protein production of the host bacteria.With clear Genetic comparison,easy culture and short fermentation period,it also has a growth characteristic when E.coli is cultured with a high density and lake of oxygen supply.E.coli host has been widely used as recombinant protein expression.E.coli using high-density fermentation is an effective way to improve the recombinant foreign gene expression,but the expression of the recombinant E.coli is affected by many factors.For the purpose of enhancing the expression of antigen,our experiments aim at optimizing the culture conditions and recombinant E.coli expression conditions,so that we can achieve high-protein expression of recombinant antigen,thus we can provide experimental data for the large-scale industrial fermentation.Methods Activating preservation's strain,using the single factor experiment and the statistical method designed experiment optimizes the different factors to fred the best expression condition.Research and measure plasmid stability,we studied the influencing factors in fermentation processes.Based on the earlier period experiment and the literature foundation,we carried on fermentation using dissolves the oxygen feedback to control strain culture.Then we used affinity chromatography to get goal antigen and identify using SDS-PAGE and Western. Result In the flask Experiments,we used response surface analysis method to optimize the best recombinant protein expression conditions based on Single-factor experiments.The best expression conditions is pH7.9,0.20 mmol/l IPTG,2.5 h duration before inducement, 5.83 h duration after inducement and 31℃for culture after inducement,a maximal expression output of 37.78 mg/L could be obtained which was two-fold of that before the optimization.Based on this we fed culture medium using dissolve the oxygen feedback method,we has obtained the OD₆₀₀value 105 and 11.08g target protein during 15h culture with a 30 L fermentation pot.we raised the output enormously,the goal protein has good immunogenic after analysis using SDS-PAGE and the Western-blot.Conclusions Through this study for optimizing the culture conditions,we have obtained the best parameters of expression,improved the production of the recombinant E.coli protein. Also we got the high cell density in the high-density fermentation of E.coli based on early experiments.These results will provide an experimental basis for large-scale industrial fermentation.Using these analytical methods,we can analyses similar studies of optimization of expression conditions.It not only can get reliable results,can also save costs and time.