莱茵衣藻BBSome蛋白BBS2原核表达、纯化和多克隆抗体的制备及鉴定

Prokaryotic Expression,Purification,Polyclonal Antibody Preparation and Identification of the Chlamydomonas reinhardtii BBSome Protein BBS2

BBSome蛋白复合物是纤毛蛋白组分之一,BBSome组装或纤毛运输缺陷可导致巴-比二氏综合征。其中,BBSome组分BBS2缺失已被临床证明是导致BBS的病因之一。莱茵衣藻是研究BBSome组装和纤毛运输的一种很好的模式生物。为深入研究BBS2的致病机制,需要特异性识别其BBS2蛋白的抗体。本文将莱茵衣藻BBS2基因5'端399 bp的cDNA序列克隆到原核表达载体pET28a-bbs2,转入大肠杆菌BL21(DE3)细胞进行诱导表达。在8 mol/L尿素存在的情况下对该融合蛋白进行镍柱亲和纯化,并将纯化后的融合蛋白免疫新西兰大白兔。SDS-PAGE电泳结果表明:分子量为15 kDa的重组6×His-BBS2融合蛋白为水不溶性蛋白。5次免疫后的抗莱茵衣藻BBS2抗血清效价高达1∶256 000。经Protein A琼脂糖CL-4B亲和纯化后的抗血清不仅在Western blotting分析中能特异性识别莱茵衣藻BBS2,而且在免疫荧光染色分析中能精确定位BBS2于基体和纤毛内。这一多克隆抗体的成功制备为深入开展BBS2在BBSome组装和纤毛运输中的分子作用机制提供了基础。

BBS2 is a BBSome subunit and its loss causes Bardet-Biedl syndrome. Chlamydomonas reinhardtii is a model organism used for studying the Bardet-Biedl syndrome. To investigate the role of BBS2 in causing Bardet-Biedl syndrome,a prokaryotic expression vector,pET28 a-bbs2 is constructed by inserting the 5’ 399 bp fragment of the bbs2 gene into the pET28 a plasmid. The recombinant vector is then transformed into Escherichia coli BL21(DE3) to induce the 6 ×His-BBS2 fusion protein expression. SDS-PAGE electrophoresis shows that this fusion protein is insoluble and expressed with a molecular weight of approximately 15 kDa as expected. In the presence of 8 mol/L urine,the fusion protein is purified by Ni-affinity purification and used to immunize New Zealand white rabbits. After the 5 th immunization,the antisera are determined to have a titer of as high as 256 000. The antisera are then purified using protein A sepharose CL-4 B.The polyclonal antibody prepared can specifically bind the C. reinhardtii BBS2 as shown in Western blotting assay and identified BBS2 in the basal bodies and in the flagellum as determined by immunofluorescent microscopy. The availability of this antibody will strengthen the research to investigate the role of BBS2 in causing Bardet-Biedl syndrome.