拟态弧菌重组蛋白OmpUa的表达条件优化及其抗原性分析

Optimization of expression conditions for recombinant outer membrane protein OmpUa of Vibrio mimicus and antigenicity analysis of OmpUa protein

目的分析重组蛋白OmpUa的表达形式和抗原性,优化拟态弧菌OmpUa基因工程重组菌(pMALc4X-OmpUa/TB1)的表达条件。方法诱导基因重组工程菌表达,分别采用SDS-PAGE和Western blot分析重组蛋白OmpUa的表达形式及其抗原性。采用正交试验设计L9(34)方案,通过SDS-PAGE和电泳图谱光密度扫描分析培养基种类、诱导温度、诱导时间和诱导剂浓度等因素对重组蛋白表达的影响。结果重组蛋白OmpUa主要以可溶性形式表达,具有良好的抗原性。基因工程重组菌接种LB培养基,37℃振荡培养3 h时加入0.5 mmol/LIPTG诱导4 h,即可获得高表达量的重组OmpUa蛋白。结论摸索出拟态弧菌OmpUa基因工程重组菌的最佳表达条件,为下一步大量制备OmpUa诊断性抗原奠定了基础。

Objective To analyse the expression form and anfigenicity of the recombinant protein OmpUa and optimize the expression conditions of gene engineering E. coli strain pMALo4X-OmpUa/TB1. Method The expression form and antigenicity of recombinant protein OmpUa induced by 0.5 mmol/L IPTG were analysed by SDS-PAGE and Weatem blot respectively. Based on the orthogonal experimental design L9 （ 34 ）, the analysis of SDS-PAGE and optical density scanning of electrophorogram, the four factors including medium component, IPTG concentration, incubation temperature and incubation time were optimized. Result The recombinant protein OmpUa with good antigenieity was expressed mainly in a soluble form. When the gene engineering E. coIi strain pMALc4X-OmpUa/TB1 was grown into LB medium, shaking incubated at 37 ℃ for 3 hours, and induced for 4 hours by 0. 5 mmol/L IPTG, a high yield of OmpUa protein of Vibrio mimicus was obtained. Conclusion The optimized expression condition of gene engineering E. coli strain pMALc4X-OmpUa/TB1 was obtained, which will be helpful for preparation of OmpUa diagnostic antigen.