重组人VEGF165 蛋白在毕赤酵母中高效表达与多克隆抗体与制备.docVIP

重组人VEGF165蛋白在毕赤酵母中高效表达与多克隆抗体的制备 王晓1,2，黄晓平1,2，周宇1,2，刁勇,2* （1：华侨大学分子药物研究院，福建 泉州362021；2：华侨大学生物医学学院，福建 泉州362021） 摘要：VEGF在大多数肿瘤中表达上调, 是肿瘤新生血管生成最重要的细胞因子，临床上VEGF的抗体可以抑制肿瘤的发展。为深入研究VEGF165的生物活性及VEGF165抗体在肿瘤治疗中的作用，本文构建pPIC9K-VEGF165分泌型载体，线性化后电击转化至GS115(his4)中，经MD平板筛选出阳性表达菌株并进行PCR验证。菌株发酵上清液经Sephadex G-25，Heparin Sepharose FFSephacryl S-100层析介质分离纯化，目的蛋白纯度达到95%，分子量约为24 KD。重组人VEGF165蛋白能够诱导HUVEC细胞增殖、提高HUVEC细胞的活性。重组人VEGF165蛋白免疫小鼠，制备多克隆抗体，间接ELISA法检测抗体效价达1：51200 。重组人VEGF165蛋白的表达及多克隆抗体的成功制备为进一步探索VEGF165在肿瘤形成中的作用以及VEGF165抗体治疗肿瘤的作用奠定了基础。 关键字： 重组人VEGF165蛋白；多克隆抗体；毕赤酵母； 中图分类号： 文献标志码：A High level expression of recombinant human Vascular Endothelial Growth Factor 165 protein in Pichia pastoris and Preparation of VEGF165 Polyclonal Antibody Wang Xiao1,2，Huang Xiao-ping1,2，1,2, Diao Yong1,2* （1Institute of Molecular Medicine，Huaqiao University，Quanzhou 362021；2School of Biomedical Sciences Huaqiao University，Quanzhou 362021 Abstract: As the single most potent angiogenesis factor, Vascular Endothelial Growth Factor (VEGF) were detected up-regulated expression in most tumors, and antibodies of which were shown to suppress tumor development clinically. In order to study the biological activity of VEGF165 and the effect of VEGF165 antibody in anti-tumor therapy, the recombinant secretory vector of pPIC9K-VEGF165 was constructed, linearized and then transformed into Pichia pastoris strain GS115 (his4) by electroporation. The positive transformants were seperated by MD plate screening and confirmed by PCR reaction. Subsequently, the rhVEGF165 protein was obtained by purification from the supernatant with Sephadex G-25 、Heparin Sepharose FF and Sephacryl S-100 gel filtration chromatographmolecular weight of 24 kD and purity of 95%, showing by SDS. Furthermore, the recombinant protein was found to have high potency of inducing HUVEC cell proliferation and improving HUVEC cell activity. The polyclonal antibody generated from rhVEGF165 immunized mouse exerts the titer of 1:51200 by indirect ELISA a