The Prokaryotic Expression Of LY3 Phosphatase Gene And The Primary Preparation Of Its Monoclonal Antibody

Objective: In order to construct the prokaryotic expression plasmid pET-28a(+)-LY3 on the basis of amplifying and cloning full length human LY3 gene, and to make monoclonal antibody of LY3 to identify basic biologic activity of it. Method: We designed the primers for amplifying the full length LY3 gene according to the LY3 gene sequence,then amplified the LY3 by PCR,digested it by double restriction endonuclease, recombined it into pET-28a(+), then transfected into BL21(DE3), added IPTG to induce its expression and detected it by SDS-PAGE and Western Blot. The recombinant LY3 protein was purified on Ni2+-NTA column under denature condition. The Balb/c mice were immunized with the purified protein to prepare the specific monoclonal antibodies(McAbs) Indirect ELISA was used to detect the titer. Result: The recombination plasmid was constructed and the fusion protein was induced by IPTG, The monoclonal antibodies anti LY3 protein were acquired after cell fusion and cloning. Conclusion: We construct and express the vector pET-28a(+)-LY3 successfully and obtain the monoclonal antibody(McAb) with the fusion protein as antigen.The successfuldevelopment of this monoclonal antibody against LY3 provides a powerfultool for the future study of LY3 .