Optimization Of High Density Fermentation Of Recombinant Escherichia Coli BL21-FMDV-B4

The production of genetic engineering vaccine was composed of culturing engineering bacteria through fermentation and purifying target protein.Due to the product cost,high density fermentation technology was always used in culturing engineering bacteria.This study was focus on optimizing high-density-fermentation technology,through optimizing culturing parameters of Escherichia coli in the ways of fermentation tank and shake bottle,the parameters including the medium,condition of culture,induce opportunity,induce times and strategy of fed-batch cultivation.Established the produce process of multiple epitopes genetic engineering Escherichia coli(BL21-FMDV-B4)vaccine of O type FMD for pigs.The results showed:1.Determined the medium formula of high-density-fermentation technology for multiple epitopes genetic engineering Escherichia coli(BL21-FMDV-B4).The medium formula(1L)was composed of peptone(22.5g),yeast powder(9.0g),glucose(1.0g),sucrose(1.0g),lactose(1.0g),inorganic salt(1.0g),amino acid(1.5g),glycerinum(0.25%)and the solution of microelement(1m L).The medium of fed-batch cultivation F1 purchased from Sichuan Bainuo jitechnology Co.,Ltd,and added 2.5% glycerinum to medium before use.2.The optimumed conditions of culture Escherichia coli(BL21-FMDV-B4)in shake bottle were:the p H value was 7.2,only 500 m L medium in bottle which volume was 2L and the concentration of inoculation was 5%.Culturing the recombinant Escherichia coli 8h,inducers were added to the medium,and the inductive time was also 8h.3.The optimumed condition of culture Escherichia coli(BL21-FMDV-B4)in 5L fermentation tank were: 37? and the p H 7.2,the concentration of inoculation was 5%.During the process of fermentation,the fed-batch cultivation method of varied exponent and constant p H was more facilitate to grow bacterium and synthesis protein.The high density fermentation of recombinant Escherichia coli(BL21-FMDV-B4)can be obtained by the optimized medium and technological parameters.The recombinant protein with high expression can be obtained.Compared with other expression systems,the Escherichia coli expression system has obvious advantages,it is equally suited for large-scale production.Multiple epitopes genetic engineering vaccine studied in this study contained the mainly neutralizing antibody epitopes of three genealogies O type FMDV,which was Type Trans Asia,Manya98 and China.In addition,epitopes of T cell were also included.This genetic engineering vaccine maybe the best candidate for substituting fully virus vaccine until now.