莱茵衣藻中IFT80蛋白多克隆抗体的制备及其应用

Polyclonal antibody’s preparation and application in chlamydomonas reinhardtii intraflagellar transport protein 80 (IFT80)

IFT80蛋白是莱茵衣藻(Chlamydomonas reinhardtii)纤毛内运输蛋白(intraflagellar transport,IFT)复合体中的一个重要组分,获得IFT80蛋白抗体为研究该蛋白作用机理提供有力的实验材料。通过构建大肠杆菌重组表达载体pGEX-4T-ift80,将重组表达载体转化到大肠杆菌E.coli Rosetta中,构建表达IFT80蛋白的重组菌株;在合适的时间点加入浓度适当的IPTG诱导重组菌株表达IFT80蛋白;将收集的蛋白上清液过柱纯化,再以纯化后的IFT80蛋白作为抗原免疫新西兰白兔,动脉取血后,将血清分离纯化获得莱茵衣藻IFT80多克隆抗体。结果:经ELISA法测定该抗体效价达到512000,Westernblot测定该抗体特异性好。该抗体可用于莱茵衣藻IFT80蛋白的检测和功能鉴定,对于研究莱茵衣藻IFT80蛋白的作用机理及生物学功能研究等具有重要意义。

IFT80 protein is one of the important components of intraflagellar transport(IFT)complex in Chlamydomonas reinhardtii,and the polyclonal antibody would provides an effective experimental material for researching the mechanism of it.Methods:The recombinant expression vector pGEX-4T- ift80 was constructed and transformed into Escherichia coli(E.coli) BL21.The recombinant strain expressing IFT80 protein was constructed,and the protein was induced by adding IPTG at the right time,and the supernatant of BL21 lysates with GST-IFT80 was purified by L-Glutathione absorption column.The purified IFT80 protein was used as antigen for immunity to New Zealand white rabbits,and the serum was isolated and purified by immunoaffinity column.Results:The antibody titer was determined to be 512000 by ELISA and can be used to detect and identify the IFT80 protein of Chlamydomonas reinhardtii precisely by Westernblot.It will be of great significance for the research for the biological function of Chlamydomonas reinhardtii IFT80 protein.