山羊c-Myc原癌基因原核表达和多克隆抗体的制备

摘要

c-Myc原癌基因与胚胎干细胞(ES细胞)和诱导的多能性干细胞(iPS细胞)生物学特性密切相关,因此制备其相应的多克隆抗体尤为必要.本研究以pMD18T-Myc质粒为模版,PCR扩增c-Myc片段,然后将其亚克隆到pSE380表达载体上,获得pSE380-Myc重组质粒.该质粒转化工程菌BL21(DE3),IPTG诱导表达His-Myc融合蛋白,随后用SDS-PAGE电泳及Western blot加以验证.在变性条件下用Ni-NTA argrose介质分离纯化His-Myc融合蛋白,并以此为抗原免疫新西兰大白兔.经过4次免疫,采集血液、分离血清,用Western blot检测抗体特异性.结果表明:(1)pSE380-Myc重组质粒在工程菌BL21( DE3)得到了高效表达；(2)得到了高纯度高含量的His-Myc融合蛋白；(3)经Wstern blot检测发现,c-Myc多克隆抗体与His-Myc融合蛋白能够特异性结合.本研究获得的特异性山羊c-Myc多克隆抗体,为进一步研究山羊ES细胞和iPS细胞的自我更新机制奠定了基础.%It is necessary to prepare polyclonal antibody of c-Myc in Capra Hircus since c-Myc protoncogene plays an important role in maintaining biological characteristics of embryonic stem cells (ES cells) and inducing pluripotent stem cells (iPS cells). The plasmid pMD18T-Myc was used as templete to amplify c-Myc fragment, which was subcloned into vector pSE380 to construct recombinant plasmid pSE380-Myc. The plasmid was then transformed into E. coli BL21 (DE3), and His-Myc fusion protein was expressed with induction of IPTG, which was subsequently verified by SDS-PAGE and Western blot assay. Purified with Ni-NTA argrose under denaturing conditon, His-Myc fusion protein was applied as antigen to immunize New Zealand White rabbits, whose blood was collected, serum (polyclonal anti-c-Myc antibody) was isolated after 4 times of immunization, and its specificity was detected with Western blot. The results showed that- (1) The recombinant plasmid pSE380-Myc was efficiently expressed in E. coli BL21 (DE3); (2) His-Myc fusion protein with higher purity was obtained; (3) Western blot analysis illustrated that the polyclonal anti-c-Myc antibody could specifically respond to His-Myc fusion protein. In conclusion, the polyclonal anti-c-Myc antibody obtained in the present study will lay a foundation for the research of self-renewing mechanism of ES cells and iPS cells in Capra Hircus.