Prokaryotic Expression Of C-Myc Gene And Preparation Of Polyclonal Anti-c-Myc Antibody And Monoclonal Anti-c-Myc Antibody In Capra Hircus

c-Myc is a important protoncogene. As a kind of transcription factors, It plays an important role in maintaining biological characteristics of embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).It is urgently necessary to prepare its polyclonal and monoclonal anti-c-Myc antibody in Capra Hircus, which will lead to further study self-renew mechanism of ES cells and iPS cells in Capra Hircus.1. The plasmid pMD18T-Myc was used as templete to amplify c-Myc fragment, which was subcloned to vector pSE380to construct recombinant plasmid pSE380-Myc.The plasmid was then transformed into Ecoli. 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 used as antigen to subcutaneously inject New Zealand white rabbits for four times at intervals of2-3weeks.7days after the last immunization, blood samples were collected, serum (polyclonal anti-c-Myc antibody) was isolated, and its specificity was detected with Western blotting assay. The results showed that (1) recombinant plasmid pSE380-Myc was expressed efficiently in E. coli BL21;(2) His-Myc fusion protein obtained in present study was of higher quality and quatity;(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 was prepared successfully in the present study.2. His-Myc fusion protein was used as antigen again to innoculate female BALB/C mice at age of7-9weeks. After4times of innoculation, spleen cells were isolated from the inoculated mice, and fused with SP2/0myeloma cells. Positive cell clones were screened with indirect ELISA, and by means of limiting dilution assay, subsequently subcloned for3-4times to obtain hybridoma cell strains that stably produce monoclonal antibody specific for c-Myc. The results showed that (1) three hybridoma cell strains were obtained (named as1F10,2H10and3B10respectively), which were able to produce stably monoclonal antibody specific for c-Myc, and the valence of antibody for their culture supernatant were1:10000,1:8000and1:6000respectively;(2) Large amount of monoclonal antibodies were prepared from ascites of BALB/C mice inoculated with hybridoma1F10and2H10, their antibody valences were1:1280000and1:640000respectively;(3)mouse monoclonal antibody isotyping showed that all of the monoclonal antibodies obtained here belonged to IgG I subclass;(4) karayotype analysis showed that the chromosome modem was90-100;(5) different passages of3bybridoma cell drains and survivals of those cryopreserved could stably produce monoclonal antibody, which was identified by indirect ELISA assay for OD values of their culture supernatant. In conclusion, monoclonal antibody specific for Capra Hircus c-Myc was successfully prepared.