人抗酶抑制因子-1的原核表达纯化及多克隆抗体的制备

摘要

We aim to carry out prokaryotic expression and purification of human antizyme inhibition factor-1(AZIN1),and to prepare as well as identify polyclonal antibody against AZIN1. Prokaryotic expression vector pET-28a/AZIN1 was transformed into Escherichia coli BL21(DE3),in which the recombinant AZIN1 was expressed by IPTG induction and purified by affinity chromatography with Ni-NTA resin under denaturing condition. The purified recombinant AZIN1 was used as the antigen to prepare the antibody in the BALB/c mice. The titer and specificity of anti-sera were detected by ELISA,Western blot,cell immunofluorescence,and immunochemistry. As results,restriction analysis and DNA sequencing proved that the plasmid pET-28a/AZIN1 was constructed successfully. In E. coli,recombinant AZIN1 protein was expressed by IPTG induction and mainly existed in a form of inclusion body. The AZIN1 protein expressed in the E. coli was effectively purified using affinity chromatography. When used as the antigen to immune mice,this protein induced the production of specific antibody against AZIN1 with serum titer of 1640000. The prepared antiserum specifically recognized and bound to the AZIN1 protein expressed in human or mouse tumor cells. And this antiserum was efficiently used in the analysis for AZIN1 by Western blot,cell immunofluorescence, and cell immunochemistry. In conclusion,the prokaryotic expression and purification of human AZIN1 protein and preparation of polyclonal antibody against AZIN1 were successfully performed in this study. These results provide a basis for further research on the roles of AZIN1 in regulating cell proliferation and in disease prevention and treatment.%原核表达纯化人抗酶抑制因子-1((antizyme inhibition factor-1,AZIN1),制备并鉴定抗AZIN1多克隆抗体.pET-28a/AZIN1表达质粒转化大肠杆菌BL21(DE3)后,IPTG诱导蛋白表达,利用Ni-NTA树脂于变性条件下亲和层析纯化人AZIN1蛋白.将重组AZIN1蛋白用作抗原免疫BALB/c小鼠以制备多克隆抗体,ELISA检测抗AZIN1抗体效价,Western bloting、细胞免疫荧光、细胞免疫化学方法检测抗体的应用.结果显示,重组pET-28a/AZIN1表达质粒经酶切及测序鉴定构建正确.细菌内重组AZIN1蛋白可被IPTG诱导表达并以包涵体的形式存在.用亲和层析法能有效纯化原核表达的AZIN1蛋白,该蛋白在小鼠体内能够诱导抗AZIN1特异性抗体产生,血清效价达到1640000.制备抗体能够特异性识别和结合人及小鼠瘤细胞中表达的AZIN1蛋白,并可有效用于AZIN1的Western blotting、细胞免疫荧光和细胞免疫化学分析.成功原核表达和纯化了人AZIN1蛋白并制备了抗AZIN1多克隆抗体,为深入研究AZIN1在调控细胞增殖及在疾病防治中的作用提供了研究基础.