真菌免疫调节蛋白在大肠杆菌中的表达及其抗体制备

Expression of fungal immunomodulate protein(LZ-8)in E.coli and its antibody preparation in rabbits

目的 通过在大肠杆菌中表达真菌免疫调节蛋白LZ-8，以制备高效价的抗体。方法 将所克隆的LZ-8基因构建成原核表达载体pET-30a—lz8，转化人大肠杆菌BL21感受态细胞中，经IPTG诱导其表达；原核表达产物经金属镍离子螯合柱纯化后，采用常规方法制备家兔多克隆抗体，并用间接ELISA法测定阳性血清的效价。结果 大肠杆菌表达产物经SDS—PAGE电泳检测到相对分子量约为14ku的目的蛋白；用考马斯亮兰法测定所表达的外源蛋白表达量为34．46mg／L；纯化后的目的蛋白免疫家兔后。经间接ELISA检测阳性血清的稀释比例达到了10^4．证明该蛋白具有良好的抗原性。结论 真菌免疫调节蛋白LZ-8能够在大肠杆菌中获得高水平的表达。且获得了高效价的家兔多克隆抗体。为以后对该蛋白的功能研究打下了基础。

Objective Through the expression of fungal immunomodulate protein （LZ-8） in E. coli to get satistactory antibody. Method The LZ-8 gene was cloned into the Vector pET-30a-lz8 and expressed in E. coli BL21, which was induced by IPTG;the total protein of induced by E. coli was purified using Ni-NTA, then the purified protein was used to make polyclonal antibody by routine method, the antigenicity was detected by ELISA. Results Above- mentioned expressed protein （about 14ku） amount to 34.46mg/L according to Coomassie Brilliant; ELISA detection showed that the protein had a satisfactory antigenicity, which was diluted about 104 fold. Conclusion Fungal immunomodulate protein （LZ-8） can be expressed in E.coli, and the high satisfactory antigenicity polyclonal antibody is obtained from rabbit, thus providing basis of following research on this protein.