Polyclonal Atibody Preparation And Subcellular Localization Of MGC5306 Protein

MGC5306 gene is a newly discovered gene, the genetic research and related reports are few in both at home and abroad.The function and mechanism of MGC5306 gene is not entirely clear. Some experimental evidence show MGC5306 gene involves in the ovarian cancer. This experiment is mainly through the related gene cloning , induced expression of recombinant proteins and antibody production methods to get people MGC5306 polyclonal antibody, the protein to facilitate our future research on MGC5306 gene's function and mechanism.We firstly get about 300bp gene segments from MGC5306 (98-204AA) gene by using the method of PCR from pEGFP-N1-MGC5306 template amplification.And then we constructed recombination plasmid through the digestion with XhoI EcoR I and lingase .we tansformed the expression of vector pGEX-4T-1-MGC5306 into escherichia coli expression BL21DE3(plysS), through the IPTG induced expression, and then SDS-PAGE electrophoresis and Western Blot testing results show that MGC5306 fusion protein GST-MGC5306 are expressed in BL21 DE3 (plysS) ,the weight of the fusion protein molecular are 36kDa. Then we identified the soluble of that protein expression which proved not dissolve ,so we cannot get purified fusion protein throngh normal affinity chromatography purification, then we use SDS-PAGE electrophoresis, the cutting objective bands, dialysis and freeze-drying obtained purified recombinant protein.Then we use the purified GST-MGC5306 fusion protein as antigens immune the white rabbits and get the serum, then using HiTrap MabSelect chromatography affinity column through the AKTA protein purification system, we get purified antibody.By ELISA test of serum antibody titer, with Western blot detection antibody specificity. Results show that we successfully construced prokaryoticexpression vector.ELISA positive and antibody titer cloning is 1:25600 and specially identify endogenous protein.Then we build eukaryotic expression vector.restructuring plasmid pDsRED-C1-MGC5306. And through Hela transfection,to check distribution and structure of the cell nucleus,we found the expression of protein located within the nucleus.