Optimization Of Culture Conditions And Genetic Stability Of Recombinant OmpA Genetically Engineered Bacteria

Escherichia coli is a common gram-negative pathogen causing mastitis in dairy cattle.In recent years,there are more and more reports about Escherichia coli causing mastitis in dairy cattle.Because of emerging of the numerous drug-resistant strains,the antibiotic treatment of dairy cow mastitis is not effective.As a result,the research and use of vaccinations to prevent and treat the disease are encouraged.Previous studies have shown that recombinant OmpA immunization induced good immune response and immune protection against Escherichia coli infection in mice,as well as cross-protection against Shigella and Klebsiella infection.So OmpA is a potential candidate antigen for vaccines.However,the genetic stability and conditions of rOmpA gene expression in engineering bacteria need to be studied,which can provide references for large-scale production of rOmpA vaccine against mastitis caused by E.coli in dairy cattle.In the experiment,the initial pH,inoculum quantity,shaking speed and IPTG concentration for inducing rOmpA expression were optimized by the single factor method during cultivation of the engineering bacteria.As a result,initial pH 7-8 in culture,inoculating medium with 1percent seedculture,shaking at 160-180 r/min,0.1 mmol/L IPTG for inducing expression in the culture of E.coli engineering bacteria were the best culture conditions.After the engineering strains were transferred to 50 generations,the culture of the 0th,5th,10 th,15th,20 th,25th,30 th,40th and 50 th generations were collected and the genetic stability of the strain was verified by morphological observation,growth situation,gene mutation and expression products.After the engineering strain transferred to 50 generations,results showed that the bacteria colony,cell size and morphology,and the growth curve were not different from the first engineering bacteria.The expression vectors of the engineering strain after passages were digested into 5900 bp and 1041 bp by double endoenzymes,the results of gene sequencing were consistent with the expression vector of the primary passage,and the results of gene sequencing were consistent with 100 in the primary passage.The molecular weight of the expressed protein was about 56 kD detected by SDS-PAGE gel electrophoresis and western blotting,which was in accordance with the initial passage of the strain.The above results show that the geneticengineering bacteria expressing rOmpA have good genetic stability.