Prokaryotic expression and polyclonal antibody preparation of the glutathione S-transferase and midkine fusion protein

VernacularTitle:谷胱甘肽-S转移酶-中期因子融合蛋白原核表达及多克隆抗体的制备
Author: Xiang GUO
Publication Type:Journal Article
Keywords: Midkine; Prokaryotic expression; Polyclonal antibody
From: Chinese Journal of Primary Medicine and Pharmacy 2012;19(6):841-843
CountryChina
Language:Chinese
Abstract: ObjectiveTo express and purify glutathione S-transferase(GST) and midkine(MK) fusion protein and prepare the ployclonsl antibody against MK.MethodsThe coding sequence of MK gene was amplified by RT-PCR from human gastric cancer tissue and inserted into pGEX-1λT vector.The recombinant vector was transformed into E.coli.BL21 ( DE3 ) to express the fusion protein GST-MK via IPTG induction.The expressed fusion protein was purified by GST affinity system and used to immunize rabbits for preparing the ployclonal antibody against GST-MK.ResultsThe expression vector pGEX-MK was constructed successfully.The expressed fusion protein was obtained by GST affinity system.The titer of the polyclonal rabbits antiserum was 1 ∶ 64 000 by indirect ELISA,Western blotting analysis showed that the antibody specifically bind to MK.ConclusionThe expressed protein and prepared polyclonal antibody provided useful reagents for MK further reaserch.