Expression And Purification Of REm18-GST Recombinant Protein And Preparation And Indentification Of Polyclonal Antiserum Against Em18 Antigen

Objective: To express recombinant pET41a-Em 18 in Escherichia. coli and optimize the purification of rEml8 protein, obstain polyclonal antiserum against Em18 antigen for the foundation of further screening for Em18 related peptides by phage display system. Methods: The recombinant expression plasmid pET41a-Em 18 was transformed into BL21(E.coli) via IPTG induction. Expressed protein was confirmed by SDS-PAGE. The expressed recombinant proteins were broken up by the different procedure of sonication and with different protease inhibitor cocktail. Recombined Eml8-GST was purified by Glutathione Sepharose-4B column, His-Bind-Ni resin and both respectively, which concentration was changed in equilibration buffer, washing buffer and elution buffer. Purified recombined protein was confirmed by SDS-PAGE. Purified Em18-GST as immunogen was used to immunize BALB/c mice. The specificity of mice anti-Em18-GST antibody was analyzed by Western blot and ELISA. Results: â‘  The expressed rEm18-GST recombinant protein can be detected as a band of 50kDa by SDS-PAGE â‘¡The high concentration of rEm18-GST was obstained by the optimized procedure of sonication: work 1s, rest 3s, and adding protease inhibitor...