Parkin蛋白C端的表达及抗体的制备

Expression of c-terminal of Parkin in E.coli and the preparation of antiserum

目的 :构建Parkin基因 3’端的表达质粒 ,在大肠杆菌中表达并制备多克隆抗体。方法 :构建Parkin基因3’端 (937～ 195 9bp)的谷氨酰胺硫转移酶 (glutathion sulfate transferase ,GST)融合表达质粒 ,在JM 10 5中获得表达。用Triton 10 0 (1% )和Tween 2 0 (1% )处理 ,并用亲和层析纯化表达产物 ,将其免疫新西兰兔 ,用免疫印迹分析收获的兔抗血清。结果 :构建的ParkinC表达质粒在JM 10 5中获得表达 ,以分子量为 4 2kD的包涵体存在 ;目的蛋白纯度为 95 % ;得到效价为 1∶6 4的抗血清 ;免疫印迹分析表明 ,制备的抗血清可与鼠脑细胞抽提物中 5 1 6kD的蛋白产生特异的免疫反应。结论 :Parkin蛋白C端在 JM 10 5

Objective To clone and express 3' terminal of Parkin gene in E.coli, and prepare its antiserum for further study. Methods The glutathion sulfate transferase (GST) fusion expression plasmid of 3' terminal of Parkin gene (937～1959 bp) was constructed and transferred to JM 105. After being treated with Triton 100 (1%) and Tween 20 (1%) and purified with affinity chromatograph, GST Parkin C was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analysed with immunoblot. Results The GST Parkin C protein was expressed in JM 105, existing in the form of inclusion body with a molecular weight of around 42 kD; The purity of GST Parkin C was up to 95%; the titer of antiserum was 1∶64; Immunoblotting showed that the prepared antiserum could react specifically with 51.6 kD protein extracted from the mouse brain. Conclusion A high level of expression of GST Parkin C is obtained in JM 105, and its antiserum can be prepared successfully.