抗VEGF单克隆抗体Fab片段在E.coli中的分泌表达

目的：在大肠杆菌中构建、表达和纯化抗血管内皮生长因子（VEGF）的Fab片段（兰尼单抗，ranibizumab），通过发酵条件的控制实现其在大肠杆菌周质和胞外的高效分泌表达，并检测其抗VEGF的活性。方法：以pET30a为质粒载体，构建了Fd链和L链前都含有OmpA信号肽、SD序列和T7 promoter的克隆载体pET30a（+）-LC-HC，转化BL21（DE3）表达菌株，并进行了培养基、温度和IPTG诱导浓度的条件优化。结果：确定Fab片段在大肠杆菌分泌表达摇瓶发酵最佳条件为：在含有1.5% Tryptone，1% Yeast Extract，0.5% Glucose，0.15% NaCl，0.1% NH₄Cl，0.08% MgCl₂·6H₂O的1L培养基的摇瓶中，按照10%的接种量，37℃摇床培养至对数生长后期（OD₆₀₀为2左右），添加0.1mmol/L IPTG诱导剂，于16℃条件下诱导表达过夜（16h左右）。用周质破菌提取分泌至大肠杆菌周质腔的Fab片段，同时用中空纤维柱浓缩发酵培养基，最后用ProteinG亲和层析柱一步纯化洗脱，经SDS-PAGE检测分析和Brandford法测蛋白浓度得出纯化的Fab抗体片段纯度在90%以上，分泌表达纯化量为0.4mg/L。以VEGF165作为结合抗原，间接ELISA分析纯化后的Fab抗体EC50=30ng/ml。继续用该培养基在3.7L体积发酵罐中进行2L体积的发酵，获得最终的菌体产率为30g/L，可亲和纯化Fab抗体量为1.94mg/L。结论：成功实现了Fab抗体片段在大肠杆菌中的高效分泌表达，且具有很高的活性，为规模化制备Fab抗体片段提供了研究依据。

The Secretory Expression of an Anti-VEGF Antibody Fab Fragment in E.coli

Object: To construct, express, purify anti-VEGF antibody Fab fragment in E.coli, and to assay its activity.Through controlling the fermentation conditions, Fab fragment can be achieved actively and largely by secreting to the periplasmic space or extracellular medium.Methods: The co-expression plasmid pET30a-LC-HC which has the OmpA signal peptide, SD sequence and the T7 promoter before Fd chain and L chain was successfully constructed.Then the plasmid was transformed into Escherichia coil BL21(DE3).The inducing condition including medium, temperature and IPTG concentration was also optimized. Results: The best shaking fermentation condition on Escherichia coil periplasmic secretory expression: in 1L XY1 medium that including 1.5% Tryptone, 1% Yeast Extract, 0.5% Glucose, 0.15% NaCl, 0.1% NH₄Cl, 0.08% MgCl₂~6H₂O, the strains were cultured with 10% inoculum at 37℃, and then added 0.1mM IPTG until the late logarithmic growth phase(OD600 was about 2) to induced overnight(about 16h) at 16℃. Except periplasmic expression, there is also a considerable part of Fab that secrete into the medium. Fab fragment which secrected into the periplasm was extracted by breaking periplasm of bacteria.At the same time, the culture medium were concentrated with the hollow fiber column, and further purified Fab by ProteinG affinity chromatography.At last, we got the purified Fab antibody fragments with more than 90% purity by SDS-PAGE analysis.The protein concentration is measured with Brandford detection method.The amount of purified Fab from periplasm and medium is up to 0.4mg/L. Taking the VEGF165 as the binding antigen, the half-maximal effective concentration (EC50) of the purified Fab is 30ng/ml being analyzed indirectly by ELISA, which show a high antigen-binding activity. Then 3.7L fermentor with 2L volume of XY1 culture mediun is used to do small pilot scale fermentation.The final cell yield is 30g/L in the 3.7L fermenter with 2L culture medium, and the concentration of Fab antibody purified by affinity column is 1.94mg/L. Conclusion: Fab fragment can be successful and high-efficiently secrected into the periplasmic space and extracellular medium through constructing the co-expression vector and optimizing the fermentation condition, and has highly activity against VEGF. It provides research basis for large-scale preparation of Fab antibody fragment.