盘基网柄菌allC的克隆、表达和多克隆抗体的制备

2010-08-21陈能星魏晓静侯连生

华东师范大学学报(自然科学版) 2010年4期

关键词：融合

陈能星, 魏晓静, 刘伟, 侯连生

(华东师范大学生命科学学院,上海 200062)

盘基网柄菌allC的克隆、表达和多克隆抗体的制备

陈能星, 魏晓静, 刘伟, 侯连生

(华东师范大学生命科学学院,上海 200062)

运用RT-PCR技术从盘基网柄菌(Dictyostelium discoideum)总m RNA中克隆到了尿囊酸酶基因(allC),该基因编码区开放读框长1 100 bp,编码的蛋白约42 kD.由于是在野生型和突变型细胞中差异表达的片段,表明该基因在盘基网柄菌多细胞发育中起到重要作用,因此将allC克隆入融合表达载体p ET-32a(+),在大肠杆菌 E.coli BL21(DE3)中进行诱导表达带有6个组氨酸标签的尿囊酸酶(ALC)融合蛋白,经镍柱亲和层析,获得了电泳纯蛋白.用纯化的融合蛋白免疫新西兰大白兔,制备多克隆抗体.EL ISA测得制备的抗ALC蛋白的多克隆抗体的效价可达1∶64 000,Western Blo tting检测证明该抗体有较强的针对ALC蛋白的专一性.这些数据表明重组质粒表达的ALC融合蛋白具有良好的抗原性,制备ALC的多克隆抗体有良好特异性和效价,能够满足针对ALC免疫印迹和细胞内定位检测等实验要求,为深入研究ALC蛋白在盘基网柄菌多细胞发育的功能作用提供了有力工具.

盘基网柄菌; 尿囊酸酶; 原核表达; 蛋白纯化; 多克隆抗体

Abstract:The coding region of allC isobtained from totalm RNA of Dictyostelium discoideum by RT-PCR.The results show that the length of allC is 1 100 bp w ith sequence analysis and coded p rotein is 42 kD.Becase it is a differentially exp ression fragments,indicating that the gene p lay an impo rtant role in multicellular development and allC was cloned into the fusion exp ression vector p ET-32a(+)with 6 His tagged and exp ressed in E.coli BL21(DE3)host cells.and then purified by Ni2+affinity chromatography column and fusion p rotein purified was used to immune the New Zealand rabbits fo r p reparing polyclonal antibody.The fusion p rotein was successfully exp ressed and polyclonal antibody was also successfully obtained.The potency of the antibody was as high as 1∶64 000.The specificity of antibody was p roved by Western Blo tting a-nalysis of exp ression p roduct of allC.These data suggest ALC fusion p rotein has the good antigenicity.The antibody w ith high titer and specificity was obtained in the satisfaction of Western blot and localization experiment request,w hich was the foundation to farther study the characters of the allantoicase p rotein and their function during Dictyostelium discoideum development.

Key words:D ictyostelium discoideum; allantoicase; p rokaryotic exp ression; p rotein purification; polyclonal antibody