Cloning and expressing of golgi protein73 gene fragment and preparation of monoclonal antibodies against the recombinant protein

Abstract

To clone and express human Golgi glycoprotein73 protein, and prepare the monoclonal antibody (mAb) against the protein. GP73 gene was amplified from HepG2 cells by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-GP73 and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot was used to detect specificity of mAbs. The prokaryotic plasmid expressing the recombinant protein was constructed, and the GP73 recombinant protein was expressed and purified. Five hybridoma cell lines that secreted anti-GP73 mAbs were obtained. 2 of 5 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with GP73 protein. The GP73 recombinant protein is highly purified and has strong antigenicity. The anti-GP73 mAbs were prepared successfully.