Prokaryotic expression and polyclonal antibody preparation of human platelet glycoprotein Ⅵ extracellular domain
- VernacularTitle:人血小板膜糖蛋白VI胞外区片段的原核表达及多克隆抗体的制备
- Author:
Chenxue QU
;
Chuanbao LI
;
Jianzhong WANG
;
Shulan WU
;
Wenhui WAN
- Publication Type:Journal Article
- Keywords:
Platelet;
Glycoprotein Ⅵ;
Polyclonal antibody
- From:
Chinese Journal of Laboratory Medicine
2003;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and purify recombinant human platelet glycoprotein Ⅵ extracellular domain and prepare the polyclonal antibody against it.Methods Human platelet glycoprotein Ⅵ extracellular domain fragment (123~268 residues) was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-3x.The recombinant plasmid was constructed and expressed in E.coli after induction with isopropyl-?-D-thiogalactopyranoside (IPTG).The fusion protein was identified by Western blot analysis after purification by affinity chromatography.Rabbits were immunized with the purified fusion protein, and the collected rabbit antiserum was evaluated by sandwich ELISA, Western blot and flow cytometry.Results The coding sequence of GPVI extracellular domain was successfully inserted into pGEX-3x.Sequencing result showed that the cloned gene was identical as reported.After induction, a Mr 42kd fusion protein was expressed and confirmed by Western blot, which was identical to that expected.The titers of the antisera were up to 1∶[KG-*2]128. Sandwich ELISA result demonstrated that the prepared antibody recognized GPVI in human platelet. Western blot and flow cytometry revealed that the prepared antibody reacted with GPVI of platelet lysate and the native GPVI on human platelet surface.Conclusions Using purified prokaryotic expressed the fusion protein as antigen, the specific antibody was elicited in the immunized animals. The prepared polyclonal antibodies react specially with GPVI on human platelet surface and can be used for further studies of GPVI.
