细粒棘球蚴谷胱甘肽s-转移酶重组抗原的高效融合表达、纯化及免疫特性的研究

目的构建细粒棘球蚴重组蛋白谷胱甘肽s-转移酶(EgGST)的表达载体,获得纯化的重组蛋白,并对其免疫学特性进行初步鉴定,为重组疫苗研制的后续工作提供依据。方法从本室已构建的重组质粒GST/pGEM-T中酶切下GST目的片段,亚克隆入表达载体pET28a,转化入大肠杆菌BL21plys进行融合表达,经His-bind树脂纯化系统小量纯化,得到纯化的重组融合蛋白作为抗原。免疫ICR小鼠,获得抗血清。通过蛋白免疫印迹试验(Western blot)和酶联免疫吸附试验(ELISA)检测重组抗原的免疫学特性。结果成功构建具有高效表达能力的基因工程菌株,并纯化了重组蛋白,纯化的重组抗原免疫小鼠的抗血清可识别天然与重组抗原,实验组血清与对照组血清抗体含量差异有统计学意义(P<0.05)。结论重组蛋白具有良好的抗原性和免疫原性。

Expression and purification of glutathion S-transferase recombinant antigen in Echinococcus granulosus and its immunogenicity

To construct the expression vector for glutathion S-transferase(GST) recombinant antigen of Echinococcus granulosa(Eg) to obtain 6 His-GST fusion protein in order to provide more information to further development of vaccines for hydatid disease,GST/pGEM-T was digested by restriction enzymes EcoIRI and XhoII to get the GST gene,which was subcloned into expression vector pET28a and then transformed to E.coli BL21plys to express the fusion protein.The recombinant protein could be expressed in quantity under induction with IPTG and purified by His-bind protein purification kit.Then,the purified recombinant protein was immunized to ICR mice in order to induce immune responses which could be detected by Western blotting and ELISA.Under these procedures,the gene engineering bacterial strain with highly efficient expression ability had been successfully constructed and the expressed recombinant protein was purified.This recombinant antigen could induce effective immune responses,in which the excellent IgG response in mice could be elicited 6 weeks after immunization with recombinant antigen.The serum antibody contents showed significant differences between the experimental and control groups of mice,suggesting that the recombinant antigen can induce efficient humoral immune responses with good immunogenicity and antigenicity.