小G蛋白BBS3b的原核表达纯化及其多克隆抗体制备

Prokaryotic expression and purification of a small GTPase BBS3b in Escherichia coli and preparation of polyclonal antibody

[目的]制备莱茵衣藻小G蛋白BBS3b的多克隆抗体。[方法]利用莱茵衣藻bbs3b基因的c DNA序列分别构建了带有GST和6×His标签的原核表达载体p GEX-2T-BBS3b和p ET-28a(+)-BBS3b并转化至大肠杆菌BL21(DE3)诱导表达,以12%SDS-PAGE鉴定。用纯化的GST-BBS3b融合蛋白免疫新西兰大白兔,采集第4次免疫后血液并分离血清,利用间接ELISA法进行效价测定,并将抗血清依次经过Protein A纯化和硝酸纤维素膜纯化,用Western Blotting检测抗体特异性。[结果]GST-BBS3b和6×His-BBS3b融合蛋白的分子量分别为46、20 k Da。用ELISA的方法测定抗血清效价为512 000,Western Blotting检测结果显示制备的多克隆抗体能够特异性识别莱茵衣藻BBS3b蛋白。[结论]制备的多克隆抗体效价为512 000,能够特异性识别莱茵衣藻中BBS3b蛋白,为利用莱茵衣藻作为模式生物进行BBS3b在纤毛信号传导中的作用机理研究奠定了基础。

[Objective] Preparation of Chlamydomonas reinhardtii small GTPase BBS3 b polyclonal antibody. [Methods]We constructed a GST-tagged and a 6 × His-tagged prokaryotic expression vector of bbs3 b gene,p GEX-2T-BBS3 b and p ET-28a（ ＋）-BBS3 b. The fusion protein was expressed in Escherichia coli and identified with 12% SDS-PAGE. After purified by affinity chromatography,the fusion protein GST-BBS3 b was used as antigen to immune rabbits to prepare polyclonal antibody. We collected the 4th immune blood and separated the serum,the titer was determined by ELISA. The antiserum was purified by the affinity purification of Protein A and nitrocellulose membrane,the specificity of polyclonal antibodies was evaluated by Western Blotting. [Results]The molecular weight of the fusion protein GST-BBS3 b and 6 × His-BBS3 b were 46 k Da and20 k Da. The result of ELISA showed that the antibody titer was about 512 000,the results of Western Blotting showed that the polyclonal antibody was recognition to the BBS3 b protein in Chlamydomonas reinhardtii. [Conclusion]We got a specificity and sensitivity polyclonal antibody,the titer was about 512 000. It laid the foundation to continue to study the functional mechanism of BBS3 b in cilia related diseases such as obesity,diabetes,polycystic kidney disease and so on.