Prokaryotic expression,purification and polyclonal antibody preparation of C terminal Helicase domain of mouse Rig-I
- VernacularTitle:Rig-I蛋白C端Helicase结构域的原核表达、纯化及其多克隆抗体制备
- Author:
Jinju LI
;
Hua REN
;
Ziqiang WANG
;
Min QIAN
;
Bing DU
- Publication Type:Journal Article
- Keywords:
Rig-I;
Helicase;
Prokaryotic expression;
Polyclonal antibody
- From:
Chinese Journal of Immunology
2000;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a highly efficacious and sensitive immunological reagent for further investigation on the retinoic acid-induced gene I (Rig-I) of mouse .Methods:The Helicase domain coding region (726-2 240 bp) of mRig-I-H was cloned into plasmid pET15b (+) to construct the recombinant plasmid pET15b(+)-mRig-I-H.Then the plasmid was transformed into E.coli BL21 for protein expression.Rabbits were immunized with electrophoresis-purified recombinant protein to obtain the polyclonal antibody against mRig-I-H.The titer of polyclonal antibody was detected by ELISA and the specificity was identified by Western blot and Immunofluorescence.Results:The recombinant protein was expressed successfully in E.coli.Western blot analysis showed that target protein was expressed with a molecular weight of 40 kD.Titer of the polyclonal antibody was about 1∶1?105 by ELISA assay.With this antibody,we could detect the expression of Rig-I in RAW 264.7 cell line by Western blot and Immunofluorescence.Conclusion:The high level expression of Rig-I Helicase domain is induced in E.coli expressing system.Anti-mRig-I-H polyclonal antibody with high titer and fine specificity could be a novel tool in future investigation of Rig-I.
