GST/MKRN1融合蛋白的表达、纯化及多克隆抗体制备  

Expression and Purification of GST/MKRN1 Fusion Protein and Preparation of Polyclonal Antibody Against GST/MKRN1

作　　者：白云秀[1] 金蕊[1] 陈皓[1] 林坚[1] 解跃华[1] 黄君健[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2006年第1期31-33,共3页Letters in Biotechnology

摘　　要：目的:利用大肠杆菌BL21(DE3)表达GST/MKRN1融合蛋白,亲和层析分离纯化目的蛋白,进行动物免疫制备多克隆抗体。方法:MKRN1cDNA全长1449bp,编码482个氨基酸残基。将其基因片段克隆到pGEX-4T-1载体上,获得pGEX-4T-1-MKRN1原核表达重组质粒,转化大肠杆菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得可溶性表达,经谷胱甘肽Sepharose4B介质填充的层析柱分离纯化蛋白,制备抗原免疫动物,得到pGEX-4T-1-MKRN1多克隆抗体。结果:ELISA结果显示血清抗体效价可达到1∶256000。通过Western免疫印迹及免疫细胞荧光分析,自制的多克隆抗体能特异地与MKRN1蛋白相互作用,可用于免疫细胞化学分析。结论:制备了效价和特异性良好的抗MKRN1多克隆抗体,经实验验证获得的免疫血清能够满足针对MKRN1的Western免疫印迹和细胞免疫组化检测的实验要求,为今后深入研究MKRN1表达的组织分布、细胞内定位及与端粒酶催化亚基(hTERT)之间相互作用的生物学意义提供了有用的实验工具。Objective： Using E.coli BL21（DE3） to express the GST-MKRN fusion protein. Affinity chromatograph is used to purify the fusion protein. Methods： The full-length MKRN1 cDNA is about 1 449 bp and encodes 482 amino acids. A cDNA fragment encoding the MKRN1 C-terminal polypeptide sequence （117～428 aa） was obtained by PCR and inserted in-framed into the vector pGEX-4T-1, so as to express a GST-MKRN1（117～428 aa） fusion protein. The resulting recombinant plasmid was transformed into the host cells BL21 （DE3） and expression of the recombinant fusion protein was induced by IPTG induction. The soluble expressed fusion protein was purified through a Glutathione Sepharose 4B affinity eolume. The purified protein was used as antigen to immune rat to prepare specific anti-MKRN muhielonal antibodies. Results： ELISA assay showed that the titer of these antibodies is up to 1：256 000. The specificity of these antibodies against MKRN1 was further confirmed by either indirect immunofluoreseenee assay or western blotting. Conclusion： The multielonal antibody prepared can interact specifically with the MKRN1 and it can also be used in immunofluoreseenee assay. With the obtainment of MKRN1 specific antibodies, it will facilitate following studies aimed to elucidating the biologic function and physiological significance of the MKRN1 gene.

关 键 词：MKRN1 端粒酶催化亚基 融合表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]