Generation and preliminary application of polyclonal antibodies against human Argonaute2 / 中国免疫学杂志

ABSTRACT

Objective:To generate rabbit polyclonal antibody against human Argonaute2 (Ago2) protein and to identify its functional characterization for determination of differential expression and cellular localization of Ago2 protein in various cell lines.Methods:DNAstar software was applied for searching the high antigenicity region of Ago2 gene sequence termed k-Ago2.Prokaryotic expressing plasmid was constructed and transformed to E.coli BL21 (DE3) to induce expression by IPTG.The fusion protein was injected into rabbits subcutaneously to produce polyclonal antibodies after purification by gel regaining.ELISA was operated to detect antibody titer.Western blot was used to identify the specificity and sensitivity of the antibodies and detect the differential expression of Ago2 protein in various cell lines.Meanwhile,immunofluorescence experiments were arranged to show cellular localization of Ago2 protein.Results:The prokaryotic expressing plasmid was constructed correctly.K-Ago2 protein was expressed and purified,and then rabbit polyclonal antibodies against Ago2 were generated after immunization with k-Ago2 protein.The titer detected by ELISA was 1∶19 000.Western blot results demonstrated the high specificity of the antibodies.Finally,we successfully observed the differential expression and cellular localization of Ago2 protein in various cell lines.Conclusion:The polyclonal antibody against Ago2 protein has been achieved successfully.It will be propitious for the intensive study of the RNAi mechanism and even profound clinical application.