高迁移率族蛋白HMGB4的表达及多克隆抗体的制备  

Prokaryotic Expression of Human HMGB4 and Generation of Its Rabbit Polyclonal Antisera

作　　者：易朵[1] 王成[1] 帅勇[1] 肖湘文[2] 赵晓蒙[1] 周畅[1] 

机构地区：[1]湖南师范大学生命科学学院,蛋白质化学与发育生物学教育部重点实验室,中国长沙410081 [2]中山大学达安基因股份有限公司,中国广州510665

出　　处：《湖南师范大学自然科学学报》2011年第2期65-70,共6页Journal of Natural Science of Hunan Normal University

基　　金：国家自然科学基金资助项目(81071696);湖南省教育厅科研资助项目(09B059);教育部留学回国人员科研启动基金资助项目((2010)609号)

摘　　要：高迁移率族蛋白B4(high mobility group box,HMGB4)是HMGB家族的新成员,研究表明其可能在睾丸发育和精子发生等方面发挥重要作用.为进一步研究HMGB4的结构、功能及与其相互作用的蛋白而进行his-HMGB4融合蛋白表达载体的构建.序列鉴定分析后,用该重组质粒转化大肠杆菌BL21(DE3),异丙基β-D硫代半乳糖苷(IPTG)诱导产生his-HMGB4融合蛋白,继而纯化获得相对分子质量约25 000的融合蛋白.将此融合蛋白免疫新西兰兔,经Western印迹和ELISA检测,制备的抗体可特异性识别HMGB4蛋白,其效价达1∶20 000.获得的高效价、高特异性的兔抗HMGB4蛋白多克隆抗体为下阶段研究HMGB4的功能奠定了基础.HMGB4 protein is a new member of HMGB （High mobility group protein B） family, which may play important role in testis development and spermatogenesis. In order to further study the structure and biological function of HMGB4 and to investigate the proteins interacted with HMGB4, his-HMGB4 fusion protein vector was constructed. His-HMGB4 fusion protein was expressed and purified in prokaryotic system, and its polyclonal anti- body was prepared. HMGB4 cDNA codon domain was amplified from human testis by RT-PCR method and recombined into pQE-N1 plasmid expressing his-tag fusion protein. After identified by the restriction enzyme digestion and sequencing, the recombinant clone was transformed into the competent expressive cells of E. coli BL21 （DE3）. His-HMGB4 fusion protein was induced by IPTG and further purified by Ni-NTA Agarosc affinity chromatography to obtain a fusion protein with molecular weight of 25 000. Rabbits were immunized with the purified protein to - pare antisera. The antibody could recognize HMGB4 protein specifically and its titer is about 1：20 000. These results confirm that the anti-rabbit HMGB4 polyclonal antibody with high affinity and specificity has been prepared successfully, and lay a foundation for our ongoing research on the specific expression of HMGB4 in human testis.

关 键 词：HMGB4 原核表达 纯化 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]