[1]王晓,黄晓平,周宇,等.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[J].华侨大学学报(自然科学版),2014,35(6):695-700.[doi:10.11830/ISSN.1000-5013.2014.06.0695]
　WANG Xiao,HUANG Xiao-ping,ZHOU Yu,et al.High Level Expression of Recombinant Human Vascular EndothelialGrowth Factor 165 Protein in Pichia pastorisand Preparation of VEGF165 Polyclonal Antibody[J].Journal of Huaqiao University(Natural Science),2014,35(6):695-700.[doi:10.11830/ISSN.1000-5013.2014.06.0695]
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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()
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《华侨大学学报（自然科学版）》[ISSN:1000-5013/CN:35-1079/N]

卷:
第35卷
期数:
2014年第6期
页码:
695-700
栏目:
出版日期:
2014-11-20

文章信息/Info

Title:
High Level Expression of Recombinant Human Vascular EndothelialGrowth Factor 165 Protein in Pichia pastorisand Preparation of VEGF165 Polyclonal Antibody
文章编号:
1000-5013(2014)06-0695-06
作者:
王晓1; 2;  黄晓平1; 2;  周宇1; 2;  刁勇1; 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
Author(s):
WANG Xiao1; 2;  HUANG Xiao-ping1; 2;  ZHOU Yu1; 2;  DIAO Yong1; 2
1. Institute of Molecular Medicine, Huaqiao University, Quanzhou 362021, China; 2. School of Biomedical Sciences, Huaqiao University, Quanzhou 362021, China
关键词:
重组人VEGF165蛋白;  多克隆抗体;  毕赤酵母;  高效表达
Keywords:
rhVEGF165 protein;  polyclonal antibody;  Pichia pastoris;  high level expression
分类号:
Q815
DOI:
10.11830/ISSN.1000-5013.2014.06.0695
文献标志码:
A
摘要:
构建pPIC9K-VEGF165分泌型载体,线性化后电击转化至GS115(his4)中,经最小葡萄糖培养基(MD平板)筛选出阳性表达菌株并进行聚合酶链式反应(PCR)验证.菌株发酵上清液经Sephadex G-25,Heparin Sepharose FF和Sephacryl S-100层析介质分离纯化,目的蛋白纯度达到95%,相对分子质量约为24 ku.结果表明:重组人VEGF165蛋白能诱导人脐静脉内皮细胞(HUVEC)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,间接酶联免疫吸附测定法(ELISA)检测抗体效价达1:51 200.
Abstract:
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 minimal dextrose medium(MD plate)plate screening and confirmed by polymerase chain reaction(PCR). Subsequently, the rhVEGF165 protein was obtained by purification from the supernatant with Sephadex G-25, Heparin Sepharose FF and Sephacryl S-100 gel filtration chromatograph with relative molecular mass of 24 ku and purity of 95%. Furthermore, the recombinant protein was found to have high potency of inducing HUVEC cell proliferation and improving human umbilical vein endothelial cell(HUVEC)cell activity. The polyclonal antibody generated from rhVEGF165 immunized mouse exerted the titer of 1∶51 200 by indirect ELISA analysis.

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备注/Memo

备注/Memo:
收稿日期: 2014-03-05
通信作者: 刁勇(1967-),男,教授,主要从事新药研发、基因药物的研究.E-mail:diaoyong@hqu.edu.cn.
基金项目: 国家自然科学基金资助项目(81271691); 国际科技合作项目(2011DFG33320); 福建省科技重大项目(2013N5007); 华侨大学科研基金资助项目(11HZR19)
更新日期/Last Update: 2014-11-20