DNA损伤检查点蛋白调节子1片段的表达纯化及抗体制备

目的：原核表达、纯化DNA损伤检查点蛋白调节子1（MDC1）片段,并制备其多克隆抗体。方法：设计特异引物,通过RT-PCR扩增编码MDC1 N端194个氨基酸残基的基因片段,测序正确后插入含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克隆抗体,用ELISA测定抗体的效价,Western印迹鉴定抗体的特异性。结果：原核表达并纯化了MDC1 N端片段,并获得了抗MDC1的多克隆抗体,抗体效价达到1∶12800,Western印迹显示该抗血清能特异识别原核及真核细胞表达的MDC1。结论：MDC1 N端片段能够诱导小鼠产生具有较高效价和特异性的多克隆抗体,为进一步研究MDC1在Fhit特异信号通路中的作用奠定了基础。

Expression and Purification of Mediator of DNA Damage Checkpoint Protein 1 Fragment and Preparation of its Polyclonal Antibody

Objective： To express and purify mediator of DNA damage checkpoint protein I（MDC1） fragment in E.coli, and prepare the polyelonal antibody against MDC1. Methods： The specific primers were used to amplify the gene coding MDC1 N-terminal fragment of 1-194 amino acids by RT-PCR, then the gene was inserted into the vector pGEX-KG containing GST gene, GST-MDC1N fusion protein were expressed in E.coli, and purified by using Glutathione Sepharose 4B. Then anti-MDC1 antiserum was prepared in mice, the titer was determined, by ELISA and the specificity was identified by Western blot. Results： MDC1N was expressed and purified, the polyclonal antibody against MDC1 was prepared, the titer of this antibody was about 1：12 800, and MDC1 expressed in prokaryotic and eukaryotic cells can be recognized by this antiserum. Conclusion： MDC1N fragment can induce the effective and specific polyclonal antibody in mice, it is the basis to understand its function in the specific Fhit signal pathways.