The immunoactivity study of purified recombinant CP<sub>4</sub>-EPSPS protein

TANG Ni; et al

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The immunoactivity study of purified recombinant CP₄-EPSPS protein

更新时间：2024-01-17

- The immunoactivity study of purified recombinant CP₄-EPSPS protein
- Journal of Chongqing Medical University Issue 1, Pages: 10-13(2006)
- 作者机构：
  
  1. 教育部感染性疾病分子生物学重点实验室重庆医科大学病毒性肝炎研究所
  2. 加拿大Artron Bioresearch Inc
  3. 教育部感染性疾病分子生物学重点实验室重庆医科大学病毒性肝炎研究所,重庆,400010
- 作者简介：
- 基金信息：
- DOI：
  CLC： R392.1
- Published：2006，
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[1]唐霓,Yu Xiang,Helen Ke,Zhihui Yao,敬凌霞,黄爱龙,郑建.重组抗原CP_4-EPSPS的表达及免疫学活性研究[J].重庆医科大学学报,2006(01):10-13.

TANG Ni, et al. The immunoactivity study of purified recombinant CP₄-EPSPS protein. [J]. Journal of Chongqing Medical University (1):10-13(2006)
[1]唐霓,Yu Xiang,Helen Ke,Zhihui Yao,敬凌霞,黄爱龙,郑建.重组抗原CP_4-EPSPS的表达及免疫学活性研究[J].重庆医科大学学报,2006(01):10-13. DOI：

TANG Ni, et al. The immunoactivity study of purified recombinant CP₄-EPSPS protein. [J]. Journal of Chongqing Medical University (1):10-13(2006) DOI：

摘要

目的:表达并纯化重组CP4-EPSP合成酶（CP4-EPSPS）

研究重组CP4-EPSPS的免疫学活性;为进一步制备单克隆抗体和开发检测CP4-EPSPS的快速诊断试剂奠定基础。方法:诱导表达含转基因大豆CP4-EPSPS的重组载体pET-EPSPS

经Ni-NTA琼脂糖树脂亲和层析纯化

以ELISA、胶体金试条、W estern杂交鉴定其免疫活性。结果:重组CP4-EPSPS以包涵体表达为主

上清表达量极低

但胶体金试纸条检测发现上清中CP4-EPSPS抗原活性强;复性包涵体的抗原性与上清的抗原性明显不同。采用扩大培养量、缩小超声破碎重悬体积的方法

从上清中纯化CP4-EPSPS获得成功

ELISA检测此重组抗原免疫小鼠血清抗体滴度可达到1:625

000;W estern杂交证实重组抗原免疫血清与CP4-EPSPS标准品有较好的免疫反应性。结论:包涵体的简单复性方法难以恢复其关键的抗原决定簇的免疫原性;运用胶体金试纸条检测重组CP4-EPSPS活性

灵敏度高

简单快捷

对CP4-EPSPS特异检测相关的关键性抗原决定簇的甄别有重要作用

可指导重组抗原的纯化和在免疫学研究、特别是在单克隆抗体开发中的应用。

Abstract

Objective: To obtain highly purified recombinant CP4-EPSPS antigen with well reserved original antigenicity by a simple and fast protocol

and to study immunoactivity of recombinant CP4 EPSPS antigen.Methods: Recombinant expression vector pET-EPSPS containing CP4-EPSPS gene from transgenetic soy bean was induced by optional concentration of IPTG.After antigen activity was identified by strip-test method

recombinant protein expressed in the supernatant was purified by Ni-NTA-chelated agarose affinity chromatography method.Immunoactivity of purified CP4-EPSPS was further confirmed by ELISA assay

strip-test method

and Western blot analysis.Results: Recombinant CP4-EPSPS antigen was mainly expressed in the form of inclusion bodies in E.Coli system

whereas the strong antigenicity of CP4-EPSPS was detected in the induced supernatant by Colloidal-Gold strip-test

no antigenicity of CP4-EPSPS could be detected by Colloidal-Gold strip test for inclusion bodies or renatured sample from inclusion bodies.CP4-EPSPS protein was successfully purified from induced supernatant via propagating host cell culture and reducing the re-suspension volume of induced culture.ELISA assay indicated the specific antibody titer of recombiant antigen-immunized serum sample could reach 1:625

000 after three months of immunization.Using serum sample derived from recombinant antigen-immunized mice as primary antibody

CP4-EPSPS standard and purified recombinant antigen showed a highly specific and strong reaction with this serum by Western blot analysis.Conclusion: The original antigenicity of CP4-EPSPS cannot be recovered completely during gradual renaturing procedures by simple dilution methods.The epitope related to the rapid detection of CP4-EPSPS cannot be recovered by renaturing the inclusion bodies by the method in this study.However

CP4-EPSPS in induced supernatant is found to have a well-reserved original antigenicity.Colloidal Gold Strip-test to detect recombinantCP4-EPSPS antigen is a highly sensitive

and convenient method to follow through the whole process of recombinant expression and purification.It plays a crucial role in identifying the induced recombinant CP4-EPSPS for purification and further application in the immunization for the monoclonal antibody development.

关键词

CP4-EPSPS快速检测亲和层析纯化

Keywords

CP4-EPSPSStrip testaffinity chromatographypurification

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Related Institution

Department of Histology and Embryology, College of Basic Medical Sciences, Chongqing Medical University

重庆医科大学基础医学院组胚教研室

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重庆医科大学病毒性肝炎研究所

Key Laboratory of Biochemistry and Molecular Pharmacolog,Chongqing Medical University

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The immunoactivity study of purified recombinant CP4-EPSPS protein

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摘要

Abstract

关键词

Keywords

references

The immunoactivity study of purified recombinant CP₄-EPSPS protein