Prokaryotic expression,purification and polyclonal antibody preparation of C terminal Helicase domain of mouse Rig-I / 中国免疫学杂志
Chinese Journal of Immunology
;
(12)2000.
Article
Dans Chinois
| WPRIM
| ID: wpr-548913
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.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
langue:
Chinois
Texte intégral:
Chinese Journal of Immunology
Année:
2000
Type:
Article
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