人Argonaute2蛋白多克隆抗体制备及初步应用

Generation and preliminary application of polyclonal antibodies against human Argonaute2

目的:制备人Argonaute2(Ago2)的多克隆抗体,鉴定其特异性,并应用该抗体检测Ago2在人各细胞系中的表达差异及细胞定位。方法:用DNAstar软件寻找抗原性高的Ago2序列区域(命名为k-Ago2),构建k-Ago2的表达质粒,转化大肠杆菌并诱导表达。融合蛋白经切胶回收纯化后免疫大白兔制备抗体。以ELISA检测抗体效价,Western blot鉴定抗体特异性及检测Ago2在细胞系中的表达差异,免疫荧光染色观察Ago2的细胞定位。结果:成功构建表达质粒,继而k-Ago2得以表达与纯化,免疫大白兔后得到Ago2多克隆抗体。ELISA检测抗体效价为1∶19 000,Western blot确定抗体具有高度特异性,并成功地用该抗体检测到Ago2在人各细胞系中的表达差异及细胞定位。结论:Ago2多克隆抗体的成功制备,对RNAi机制的深入研究及其进一步的临床应用均具有重要价值。

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 soft-ware was applied for searching the high antigenicity region of Ago2 gene sequence termed k-Ago2. Prokaryotic expressing plasnfid 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.