构建噬菌体展示抗体库过程中电穿孔法的条件优化

Optimization of Electroporation Conditions in Construction of Phage Display Antibody Library

目的:确定稳定且高效的电转化实验方案以达到构建高库容噬菌体展示抗体库的目的。方法:探索电压、脉冲时间、噬菌粒DNA质量与浓度、TG1大肠杆菌的生长周期、重悬洗涤缓冲液及培养基优化等方面对TG1大肠杆菌电转化效率的影响。结果:当电转杯电极间距为2mm时,设定电转仪参数为3kV、25μF、5ms、200Ω;外源DNA经纯化后加入感受态菌液中使终浓度为1ng/μl;培养基中加入20mmol/L的MgCl2,并将TG1的生长阶段调控在OD600=0.8,用无菌超纯水重悬及洗涤细胞,将感受态细胞浓度调整为4×10^10个/ml。在上述条件下,电转化效率可达到4.9×10^9CFU/μg DNA。结论:通过多种条件优化,提高了电转化效率,为构建高库容噬菌体展示抗体库建立了基础。

Objective:To determine a stable and efficient electroporation protocol to construct a highcapacity phage display antibody library.Methods:Explored the effects of voltage,pulse time,quality and concentration of phagemid DNA,growth phase of TG1 E.coli,buffer for resuspension and washing and medium optimization on the electrotransformation efficiency of TG1 E.coli.Results:Using electroporation cuvettes with electrode spacing of 2 mm,the parameters of the electrorotator were set to 3 kV,25μF,5 ms,and 200Ω.The exogenous DNA was purified and added to the competent bacterial suspension to make the final concentration of DNA 1 ng/μl.20 mmol/L MgCl2 was added to the medium,and the growth phase of TG1 was adjusted to OD600=0.8.The cells were resuspended and washed with sterile ultrapure water,and the concentration of competent cells was adjusted to 4×10^10cells/ml.Under such conditions,the electrotransformation efficiency can reach 4.9×10^9 CFU/μg DNA.Conclusion:Electrotransformation efficiency has been improved through multiple condition optimization,which established the basis for constructing a high-capacity phage display antibody library.