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.
ABSTRACT
Objective To investigate the role of Kit signal pathway in the proliferation and spontaneous rhythmic motility of interstitial cells(ICs)in the bladder of guinea pigs.Methods Fourteen Guinea pigs aged 45-60 days were administered with Imatinib intragastrically for 20 and 40 days.Three were fed on normal saline as control.The morph and number of ICs were observed by immunofluorescence,Kit protein expression was detected by Western blot and spontaneous rhythmic motility of bladder were investigated in in vitro organ bath.Results Quite a few ICs were observed in the bladder of controls.After administration of Imatinib,the amount of ICs and the expression of Kit protein decreased gradually,and the spontaneous rhythmic contraction weakened.Conclusion ICs might be the pacemaker of bladder in guinea pigs.Kit protein might play an important role in the survival and functional maintenance of ICs.