基于响应面分析方法的钩端螺旋体三联属特异性蛋白抗原原核表达条件的优化

Optimization of prokaryotic expression conditions of Leptospira interrogans trigeminy genus-specific protein antigen based on surface response analysis

摘要:

脂蛋白LipL32和LipL21及穿膜蛋白OMPL1是问号钩端螺旋体（简称钩体）属特异性表面抗原，可作为通用型钩体基因工程疫苗抗原。【目的】在我们的研究中，为了获得使得重组人工融合基因lipL32/1- lipL21-ompL1/2获得较高的表达水平，我们优化了重组宿主大肠杆菌的表达条件。【方法】我们采用了基于中心复合设计（CCD）的响应面分析方法（RSM）。影响重组大肠杆菌生长的主要因素包pH、诱导剂IPTG浓度、诱导前培养实践、诱导时间以及诱导温度。响应面分析方法可以从这些因素中找到适合目的蛋白表达的最优表达条件。【结果】我们的结果显示在pH7.9、0.20 mmol/L IPTG、诱导前培养时间2.5 h、诱导时间5.83 h、诱导后温度31 ℃条件下，可获得目的重组蛋白 37.78 mg/L的最大表达量。【结论】实验结果表明采用基于中心复合设计的响应面分析方法能够较大地提高目的产物的产量。利用统计方法不但可以减少试验的次数，而且还能够从影响蛋白的因素中找出最重要的影响因素，因此该方法不仅对重组蛋白表达条件的优化有用，而且是一种很好的筛选方法。

Abstract:

Lipoproteins LipL32 and LipL21 and transmembrane protein OMPL1 have been confirmed as the superficial genus-specific antigens of Leptospira interrogans, which can be used as antigens for developing a universal genetic engi-neering vaccine. [Objective] In order to obtain high expression of an artificial fusion gene lipL32/1-lipL21-ompL1/2, we optimized prokaryotic expression conditions. [Methods] We used surface response analysis based on the central compos-ite design to optimize culture conditions of a new antigen protein by recombinant Escherichia coli DE3.The culture con-ditions included initial pH, induction start time, post-induction time, Isopropyl b-D-thiogalactopyranoside (IPTG) con-centration, and temperature. [Results] The maximal production of antigen protein was 37.78mg/l. The optimal culture conditions for high recombinant fusion protein was determined: initial pH 7.9, induction start time 2.5 h, a post-induction time of 5.38 h, 0.20 mM IPTG, and a post-induction temperature of 31℃.[Conclusion] Surface response analysis based on CCD increased the target production. This statistical method reduced the number of experiments required for optimization and en-abled rapid identification and integration of the key culture condition parameters for optimizing recombinant protein expression.