为了制备草鱼重组TAB1蛋白（rCiTAB1）及其特异性抗体，首先以草鱼头肾组织cDNA为模板，PCR扩增CiTAB1基因全长序列，并依次构建重组克隆质粒pMD19-T- CiTAB1与重组表达质粒pET-32a-CiTAB1。该重组表达质粒经0.5 mmol·L-1 IPTG，37℃诱导表达12 h后获得以包涵体形式表达的重组CiTAB1蛋白（rCiTAB1）。然后，采用3种不同方法对包涵体蛋白进行变性，发现与高浓度尿素直接变性法和洗涤后尿素变性法相比，洗涤后梯度尿素变性法处理后的蛋白纯度最好，经透析复性后得到浓度为2 mg·mL-1的rCiTAB1。最后，将rCiTAB1蛋白与白油佐剂及免疫增强剂混合，室温下混合1.5 h乳化成免疫原，3次免疫新西兰大白兔制备兔抗CiTAB1抗体，经间接ELISA方法和免疫琼脂双向扩散试验测得免疫后第33天血清中特异性抗体的效价分别为1∶1 048 576和1∶16。Western blot检测到一条分子量约为72 kDa的特异性条带，表明该抗体能特异性识别rCiTAB1蛋白。研究结果为后续深入研究CiTAB1蛋白功能提供了物质基础。
In order to prepare the recombinant TAB1 protein of Ctenopharyngodon idellus (CiTAB1) and its specific antibodies, firstly, the full-length CiTAB1 gene was amplified using the cDNA of grass carp head kidney as template in the study. Meanwhile, the recombinant cloning plasmid pMD19-T-CiTAB1 and expression plasmid pET-32a-CiTAB1 were successively constructed. Subsequently, the recombinant expression plasmid was induced by 0.5 mmol·L-1 IPTG for 12 h at 37 °C, and the recombinant CiTAB1 protein (rCiTAB1) expressed as the form of inclusion body was obtained. Secondly, denaturation of rCiTAB1 protein was carried out by using three different methods, respectively. It was found that rCiTAB1 protein treated by gradient urea denaturation after washing was purer compared with the direct high concentration urea denaturation and urea denaturation after washing, and the protein concentration reached 2 mg·mL-1 after dialysis renaturation. Finally, rCiTAB1 protein mixed with white oil adjuvant and immunopotentiator, respectively, and then stirred at room temperature for 1.5 h to obtain a stable immunogen. New Zealand white rabbits were immunized with the above-mentioned immunogen three times for preparing rabbit anti-CiTAB1 antibodies. The titers of specific antibodies in the serum at 33 days post-vaccination were 1∶1 048 576 and 1∶16 detected by indirect ELISA and immunoagar bidirectional diffusion test, respectively. A specific band with a molecular weight of approximately 72 kDa was detected by Western blot, indicating that the antibody could specifically recognize the rCiTAB1 protein. The results of this study provide the basis for the further reseach on the function of CiTAB1 protein.