人WWP2蛋白的重组表达和多克隆抗体的制备

Recombinant expression and polyclonal antibody preparation of human WWP2

[目的]克隆、表达并纯化人E3泛素连接酶蛋白WWP2的部分肽段(aa 324-517),制备兔源抗WWP2多克隆抗体并初步鉴定。[方法]PCR法从人胚肾细胞HEK-293T中扩增WWP2蛋白部分肽段的编码序列并构建原核表达质粒,大肠杆菌中诱导表达;GST亲和层析法纯化后进行TEV酶切,免疫新西兰兔制备多克隆抗体;间接ELISA、Western Blot、免疫荧光等方法检测抗体的灵敏度和特异性。[结果]构建了原核表达质粒pGEX-GST-WWP2(aa 324-517),原核表达并纯化了该重组蛋白。间接ELISA测定抗体效价可达1∶100 000以上,Western Blot检测该抗体可特异性识别体外纯化的WWP2蛋白和细胞内源性WWP2蛋白,细胞免疫荧光可检测到内源性WWP2蛋白。[结论]成功克隆、表达与纯化WWP2蛋白的部分肽段,制备出抗WWP2蛋白的多克隆抗体,可用于WWP2的免疫印迹和细胞免疫荧光分析。

[Objective] To express and purify a recombinant truncate of human protein WWP2（aa 324-517）,prepare polyclonal antibodies against WWP2 protein and test its preliminary application for further study.[Methods]WWP2 c DNA coding sequence was amplified by PCR from HEK-293 T cells and cloned into prokaryotic expression vector pGEX-4T-1.The recombinant protein GST-WWP2（aa 324-517） was expressed in E.coli BL21（DE3） under IPTG induction,then purified by GST-tag beads purification kit and digest with TEV enzyme.Immunize rabbit twice for preparation of anti-WWP2 polyclonal antibody,whose titer was measured by indirect ELISA.The antibody was subjected to Western Blot and immunofluorescence to detect its specificity and application.[Results] The prokaryotic expression plasmid pGEX-GST-WWP2（aa 324-517） was successfully constructed,then protein WWP2（aa 324-517） was expressed and purified.Indirect ELISA results showed the titer of anti-sera was above 1∶ 100 000,Western Blot and Immunofluorescence showed this WWP2 polyclonal antibody of good specificity,which can recognize both prokaryotic purified and endogenous WWP2 protein.[Conclusion] Successfully cloned WWP2 coding sequence,expressed and purified the recombinant truncate of human protein WWP2（aa 324-517）,obtained a rabbit polyclonal antibody of high quality against WWP2 protein,which could be applied in Western Blot and Immunofluorescence.