The immunoactivity study of purified recombinant CP_4-EPSPS protein
- VernacularTitle:重组抗原CP_4-EPSPS的表达及免疫学活性研究
- Author:
Ni TANG
- Publication Type:Journal Article
- Keywords:
CP4-EPSPS;
Strip test;
Affinity chromatography;
Purification
- From:
Journal of Chongqing Medical University
1987;0(01):-
- CountryChina
- Language:Chinese
-
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.
