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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。
OBJECTIVE: To clone the restin gene, which encodes 219 amino acids, from the HL60 cell line induced by retinoic acid and to construct a prokaryotic expression vector to prepare antiserum for preliminary application. Methods: The coding region of restin was amplified by PCR and the cDNA of leukemia cells induced by retinoic acid was used as a template. This was cloned into an E. coli expression vector. Restin-expressing proteins were obtained after temperature induction. New Zealand white rabbits were immunized with the target protein purified by SDS-PAGE to prepare the antisera of the protein. Using indirect immunofluorescence, the expression of restin in COS-7 cells was observed. Results: Mr of the restin protein expressed in E. coli was 26,000. After its initial purification, the antiserum prepared from New Zealand white rabbits was immunized and its titer was 1: 800 by Western blot. Indirect immunofluorescence showed that the restin protein mainly distributed in the nucleus. Conclusion: The successful preparation of anti-restin antibody provides a precondition for further study of the expression of restin protein in tissues, intracellular sub localization and signal transduction pathway.