重组抗原CP4-EPSPS的表达及免疫学活性研究

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、Western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；Western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。

The immunoactivity study of purified recombinant CP4-EPSPS protein

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.