Expression, purification and identification of recombinant platelet-activating factor acetylhydrolase fusion protein
- Author:
Ying ZOU
1
Author Information
1. Department of Dematology
- Publication Type:Journal Article
- Keywords:
Acetylhydrolase;
Gene expression;
Platelet activating factor;
Purification
- From:
Academic Journal of Second Military Medical University
2010;28(5):504-507
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To obtain the full-length of platelet-activating factor acetylhydrolase (PAF-AH) gene and express it in prokaryocytes. Methods: The full-length PAF-AH gene was amplified from peripheral blood mononuclear cells (PBMCs) by RT-PCR and then cloned into the expression vector pGEX-4T-3. The PAF-AH-GST was expressed in E. coli BL21 and purified with GST purifying system. The products was identified by SDS-PAGE and Western blot, and their enzymatic activities were detected. Results: Sequence analysis indicated that the sequence of the obtained PAF-AH gene was identical with that in the GenBank. SDS-PAGE and Western blot confirmed the successful expression and purification of PAF-AH, with the molecular weight as expected. Western blotting showed that the purified protein could be recognized by specific antibodies and had immune activities. Enzymatic activity detection confirmed that the protein could specifically hydrolyzing PAF. Conclusion: Recombinant PAF-AH protein with biological activity has been efficiently expressed in E. coli in the present study.
