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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 analyze the expression and antigenicity of recombinant OmpUa, and to optimize the expression conditions of OmpUa genetically engineered recombinant bacterium (pMALc4X-OmpUa / TB1). Methods Recombinant E. coli were induced to express in E. coli. The expression of OmpUa and its antigenicity were analyzed by SDS-PAGE and Western blot respectively. The orthogonal design was used to design L9 (34) protocol. The effects of culture medium, induction temperature, induction time and concentration of inducer on the expression of recombinant protein were analyzed by SDS-PAGE and optical density scanning. Results The recombinant protein OmpUa was mainly expressed in soluble form and had good antigenicity. Recombinant E. coli were inoculated with LB medium and cultured at 37 ℃ for 3 h. The recombinant OmpUa protein was overexpressed by adding 0.5 mmol / L IPTG for 4 h. Conclusion The best expression conditions of OmpUa genetically engineered recombinant strains of V. mimicus were found out, which laid the foundation for mass production of OmpUa diagnostic antigen in the next step.