Abstract:

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.