草鱼TAB1蛋白的原核重组表达及其抗体制备

Prokaryotic recombinant expression of TAB1 protein of Ctenopharyngodon idellus and its antibody preparation

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c DNA为模板,PCR扩增Ci TAB1基因全长序列,并依次构建重组克隆质粒p MD19-T-Ci TAB1与重组表达质粒pET-32a-Ci TAB1。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r Ci TAB1。最后,将rCiTAB1蛋白与白油佐剂及免疫增强剂混合,室温下混合1.5h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r Ci TAB1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(Ci TAB1) and its specific antibodies, firstly, the full-length Ci TAB1 gene was amplified using the cDNA of grass carp head kidney as template in the study. Meanwhile, the recombinant cloning plasmid p MD19-T-Ci TAB1 and expression plasmid p ET-32 a-Ci TAB1 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 Ci TAB1 protein(r Ci TAB1) expressed as the form of inclusion body was obtained. Secondly, denaturation of r Ci TAB1 protein was carried out by using three different methods, respectively. It was found that r Ci TAB1 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, r Ci TAB1 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-Ci TAB1 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 r Ci TAB1 protein. The results of this study provide the basis for the further reseach on the function of Ci TAB1 protein.