论文部分内容阅读
目的研究产肠毒素大肠杆菌不耐热肠毒素B亚单位(LTB)的免疫原性。评价肠道病毒71型(EV71)衣壳蛋白VP1联合LTB的鼻腔免疫效果。方法构建重组质粒pET32a-LTB,表达的6×His-LTB融合蛋白用Ni2+-NTA树脂纯化后免疫新西兰大白兔制备抗血清,ELISA检测抗血清效价。用纯化的EV71 VP1、LTB联合EV71 VP1、生理盐水分别经鼻腔免疫BALB/c小鼠,收集血清、肺和小肠黏膜冲洗液,采用间接ELISA检测IgG和分泌性IgA(sIgA)。结果已成功构建重组质粒pET32a-LTB。表达的6×His-LTB融合蛋白主要以包涵体形式表达,相对分子质量(Mr)约为30 000,免疫兔所制备的抗血清效价为1∶125 000。EV71 VP1联合LTB组的小鼠特异性IgG和sIgA水平较EV71 VP1单独免疫组和对照组有明显增高。结论在E.coli BL21(DE3)中成功表达了6×His-LTB融合蛋白,纯化的融合蛋白具有良好的免疫活性和佐剂活性。通过滴鼻免疫,LTB可有效增强特异性血清抗体反应,诱导黏膜免疫应答。
Objective To study the immunogenicity of enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit (LTB). Evaluation of nasal immunity of enterovirus 71 (EV71) capsid protein VP1 in combination with LTB. Methods Recombinant plasmid pET32a-LTB was constructed. The expressed 6 × His-LTB fusion protein was purified with Ni2 + -NTA resin and immunized New Zealand white rabbits to prepare antiserum. The antiserum titer was determined by ELISA. BALB / c mice were immunized with purified EV71 VP1, LTB combined with EV71 VP1 and normal saline respectively. Serum, lung and intestinal mucosa were collected and IgG and secreted IgA (sIgA) were detected by indirect ELISA. Results The recombinant plasmid pET32a-LTB was successfully constructed. The expressed 6 × His-LTB fusion protein was mainly expressed in inclusion bodies, with a relative molecular mass (Mr) of about 30 000. The antiserum titer prepared by the immunized rabbits was 1:125 000. EV71 VP1 combined with LTB group mice specific IgG and sIgA levels than the EV71 VP1 immunization group and the control group were significantly higher. Conclusion The 6 × His-LTB fusion protein was successfully expressed in E. coli BL21 (DE3). The purified fusion protein has good immunocompetence and adjuvant activity. By intranasal immunization, LTB effectively enhances specific serum antibody responses and induces mucosal immune responses.