碱性MAGE家族新成员restin在大肠杆菌中的表达和抗体制备  

Cloning and expression of a novel gene restin and preparation of antisera (against) the recombinant restin

作　　者：路凡[1] 杨辉[2] 王孝功[2] 蒲勤[2] 李毅[2] 赵文明[1] 赵忠良[2] 

机构地区：[1]西安交通大学生命科学院,陕西西安710048 [2]第四军医大学生物化学与分子生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2005年第3期309-311,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(No.30271457)

摘　　要：目的:从维甲酸诱导的白血病细胞系HL60中,克隆编码219个氨基酸的新基因restin,构建原核表达载体、制备抗血清并进行初步应用。方法:采用PCR技术,以维甲酸诱导的白血病细胞的cDNA为模板,扩增出restin的编码区。将其克隆入大肠杆菌表达载体中。经过温度诱导获得restin表达蛋白。利用SDS-PAGE纯化的目的蛋白免疫新西兰大白兔,制备该蛋白的抗血清。以该抗体做间接免疫荧光,初步观察restin在COS-7细胞内的表达分布。结果:大肠杆菌中表达的restin蛋白的Mr为26000。将其初步纯化后免疫新西兰白兔制备的抗血清,用Westernblot检测其效价为1∶800。间接免疫荧光显示,restin蛋白主要分布在细胞核内。结论:成功地制备抗restin的抗体,为进一步研究restin蛋白在组织中的表达、细胞内亚定位以及信号转导通路提供了前提条件。AIM: To clone a new human gene, restin, from retinoic acid-treated promyelocytic cell line HL-60, express the protein in E.coli and prepare the antisera against the protein. METHODS: The restin gene was amplified from retinoic acid-treated promyelocytic cell line HL-60 by RT-PCR and cloned into a prokaryotic expression vector. The recombinant restin was induced to express in E.coli by temperature. After preliminary purification by SDS-PAGE, the restin protein was used to immunize rabbits to obtain antisera. The titers and specificity of the rabbit anti-restin antisera were tested by Western blot and immunofluorescence analysis. RESULTS: Recombinant restin with M_r being about 26 000 was highly expressed in E.coli. The titers of the anti-sera to restin ranged from 1∶100 to 1∶800. Immunofluorescence analysis showed that restin distributed mainly in the nuclei of COS-7 cells. CONCLUSION: We successfully prepared the antisera against restin, which are useful for further investigation of biological functions of restin.

关 键 词：细胞静息相关因子(restin) 抗血清 表达 间接免疫荧光 

分 类 号：Q786[生物学—分子生物学]