Expression of GST fusion proteins of human cytochrome P 2B6 and preparation of anti-cytochrome P2B6 polyclonal antibody
- VernacularTitle:人细胞色素P450 2B6的GST融合蛋白及其抗体的制备
- Author:
Xiaojie LIN
;
Jianhong LUO
;
Yingnian YU
- Publication Type:Journal Article
- Keywords:
Cytochrome P-450;
Recombinant, fusion proteins;
Antibodies
- From:
Chinese Journal of Pathophysiology
1986;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To get large amounts of pure antigens to raise specific antibodies and to perform quantifications.METHODS: CYP2B6 (cytochrome P) cDNA fragments was ligated into BamHI restricted PGEX-3b to generate recombinants PGEX/2B6. We identified recombinants PGEX/2B6 by EcoRI digestion. The expression of fusion proteins were induced by adding isopropyl-thiogalactoside(IPTG). Several clones showed high-level expression of fusion proteins. Insoluble proteins was isolated from the bacteria and the fusion proteins was recovered and purified from a preparative (2mm) SDS-PAGE. The polyarcrylamide gel containing the fusion proteins glutathione S-transferase(GST-2B6) were used to immunize BALB/C mice from which polyclonal ascites fluid was prepared. The purified fusion proteins GST-1A1(GST fusion protein of CYP1A1 cDNA246～386aa expressed in this library ,purified by preparative SDS-PAGE), GST-2B6 were used to test the specificity of 2B6pAb. RESULTS:Fusion proteins constructed between GST and CYP2B6 was expressed in Escherichia coli DH5?. Mouse antibodies are raised against the fusion proteins GST-2B6. 2B6pAb was fond to be specific antibody.CONCLUSION:Recombinant PGEX/2B6 were constructed and purified fusion proteins GST-2B6, and specific 2B6pAb were obtained.
