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目的利用同源重组技术将柯萨奇病毒A16型(Coxsachievirus A16,CA16)VP1基因重组入枯草芽孢杆菌基因组,进而将VP1蛋白展示在芽孢表面制备CA16粘膜疫苗。将疫苗滴鼻方式免疫小鼠,检测小鼠血清中VP1特异性抗体滴度,体外中和实验检测抗体的保护作用。方法将枯草芽孢杆菌CotB基因与VP1基因连接后插入整合质粒pDG1662得到重组质粒pDG1662-CotB-VP1,电转化法将线性化重组质粒转化枯草芽孢杆菌1A771,抗生素抗性筛选,PCR、Western-Blot、免疫荧光鉴定VP1基因重组及蛋白表达情况。将该疫苗滴鼻免疫小鼠,ELISA法检测血清抗体滴度,体外中和实验分析该抗血清的中和活性。结果 VP1基因成功重组入枯草芽孢杆菌基因组中并将蛋白展示在芽孢表面。该疫苗免疫小鼠后有效的诱导了小鼠体液免疫反应,并且该抗血清在体外中和实验中表现了较好的中和活性。结论 CA16 VP1蛋白展示在枯草芽孢杆菌芽孢表面制备的疫苗能刺激小鼠产生具有保护作用的中和抗体,为研制CA16粘膜疫苗奠定了基础。
Objective To construct recombinant CA16 mucosal vaccine by subcloning the VP1 protein of Bacillus subtilis into the Bacillus subtilis genome using homologous recombination technique. The mice were immunized intranasally with the vaccine and the titer of VP1 specific antibody in the serum of the mice was detected. In vitro neutralization assay was used to detect the protective effect of the antibodies. Methods The recombinant plasmid pDG1662-CotB-VP1 was inserted into the integrated plasmid pDG1662 after the CotB gene of Bacillus subtilis was ligated with the VP1 gene. The linearized recombinant plasmid was transformed into Bacillus subtilis 1A771 by electroporation, antibiotic resistance screening, PCR, Western-Blot, Immunofluorescence identification VP1 gene recombination and protein expression. The vaccine was intranasally immunized mice, serum antibody titers were measured by ELISA, and the neutralization activity of the antisera was analyzed by in vitro neutralization assay. Results The VP1 gene was successfully recombined into Bacillus subtilis genome and displayed on the surface of spores. The vaccine effectively induced humoral immune response in mice, and the antiserum showed good neutralizing activity in vitro neutralization experiments. Conclusions CA16 VP1 protein shows that the vaccine prepared on the surface of Bacillus subtilis spores can stimulate mice to produce protective neutralizing antibody, which lays a foundation for the development of CA16 mucosal vaccine.