血管内皮生长因子全长抗体在毕赤酵母中的表达与活性鉴定

目的:构建血管内皮生长因子(VEGF)全长抗体表达载体pPICZαA-VH-CH-VL-CL,评价其在毕赤酵母中的表达产物与抗原的结合特性,及其抑制细胞增殖的活性。方法:利用基因合成分别获得VEGF抗体CL和CH序列,分别构建pPICZαA-CH和pPICZαA-CL重组质粒,再利用同尾酶特性构建双启动子表达盒的重组pPICZαA-CH-CL载体,用Westen印迹对其进行表达鉴定后将VH和VL序列插入该载体,获得VEGF的全长抗体表达载体pPICZαA-VH-CH-VL-CL;通过膜筛和ELISA进行菌株筛选,并对VEGF抗体表达阳性菌株进行小量表达和纯化,采用CCK-8法对其抑制人脐静脉内皮细胞(HUVEC)增殖的活性进行初步评价。结果:获得表达轻、重链的VEGF抗体表达载体,ELISA实验证明pPICZαA-VH-CH-VL-CL具有一定的VEGF抗原结合特性;体外增殖实验表明,该抗体可以以剂量依赖性抑制HUVEC增殖。结论:在毕赤酵母中表达、纯化了具有一定功能活性的VEGF全长抗体,为后续比较研究酵母糖基化改造对VEGF抗体的药效学和药代学的影响提供了基础。

Expression of VEGF Full Length Antibody in Pichia pastoris and its Activity Detection

Objective： To construct and express the full-length human vascular endothelial growth factor（VEGF） antibody in Phchia pastoris yeast and evaluate the antibody characteristic and ability to inhibit human umbilical vein endothelial cells（HUVEC） growth. Methods： VEGF constant region genes of the heavy and light chains（CH and CL） antibody sequences were synthesized and were cloned to pPICZαA, respectively, to make pPICZαA-CH and pPICZαA-CL. Then we constructed the double promoter series expression vector pPICZαA-CH-CL using iso-caudarner BglⅡ and BamHⅠ. The expressed product were identified by Western blot and full-length human VEGF antibody-expressed vector, pPICZαA-VH-CH-VL-CL, were constructed by inserting the VEGF varient re-gion genes of the CL and CH into pPICZαA-CH-CL. Blotting and ELISA were performed to screened the optimal clone. After purification by Nickel affinity purification column, the purified proteins were used to evaluate the HU-VEC growth by CCK-8 assay. Results： The full-length human VEGF antibody pPICZαA-VH-CH-VL-CL was suc-cessfully constructed and expressed by sequencing and Western blot. ELISA showed that the VEGF antibody has affinity with the VEGF antigen. In addition, the purified VEGF antibody could inhibit cell growth in dose manner. Conclusion： We successfully constructed and expressed the functional full-length human VEGF antibody in P.pas-toris yeast, which lay establishment to the comparison study of VEGF antibody pharmacokinetics and pharmacody-namics in wild and N-glycoengineered P.pastoris.