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

目的 构建谷胱甘肽-S-转移酶(GST)和中期因子(MK)融合蛋白的原核表达质粒,并表达和纯化蛋白,制备多克隆抗体.方法 通过RT-PCR技术从人胃癌组织中扩增入MK编码序列,克隆入表达载体pGEX-1λT中,获得表达质粒pGEX-MK,并在大肠杆菌BL21 (DE3)中经IPTG诱导表达,通过亲和层析纯化表达的GST-MK融合蛋白,并以重组蛋白免疫兔子.结果 成功构建了GST-MK融合蛋白的原核表达载体,经诱导表达纯化得到GST-MK融合蛋白.免疫兔子后取多抗血清以间接ELISA检测效价达1∶64 000,Western blotting分析显示多克隆抗血清对MK蛋白特异结合.结论 MK在大肠杆菌中成功表达及其多克隆抗体的获得,为研究MK生物功能奠定了基础.

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

Objective To express and purify glutathione S-transferase（GST） and midkine（MK） fusion protein and prepare the ployclonal antibody against MK. Methods The coding sequence of MK gene was amplified by RT-PCR from human gastric cancer tissue and inserted into pGEX-IkT vector. The recombinant vector was transformed into E. coll. 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. Results The 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. Conclusion The expressed protein and prepared polyclonal antibody provided useful reagents for MK further reaserch.