碱性MAGE家族新成员restin在大肠杆菌中的表达和抗体制备

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。

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

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.