预防性重组HPV58-减毒志贺氏杆菌载体活疫苗的研制及免疫学特性

目的：构建预防性重组HPV58-减毒志贺氏杆菌载体活疫苗，并研究其免疫学特性。方法：克隆HPV58L1基因并重组入自杀载体pCVD442，减毒志贺氏杆菌Sf301：△virG、辅助质粒PRK2013、重组自杀载体pCVD442-HPV58L1进行筛选杂交，经氨苄抗性培养基、刚果红培养基双重选择，获得重组的含HPV58L1基因的减毒志贺氏杆菌菌株。Western blot检测HPV58L1蛋白表达。用豚鼠角结膜炎模型进行疫苗动物免疫试验，ELISA检测豚鼠血清中特异性抗体，ELISPOT检测豚鼠脾及淋巴结中抗原特异性IgG、Iga产生细胞频数。结果：Western blot法证实该菌株可表达HPV58L1蛋白。豚鼠免疫保护试验发现：HPV58L1-减毒志贺氏杆菌不引起角结膜炎，经用野生型sf301攻击后有80％豚鼠不发病。免疫后第20天，免疫豚鼠血中抗HPV58L1特异性IgG、IgA明显升高，而对志贺氏杆菌菌体抗原（LPS）仅产生低效价的IgG抗体。ELISPOT结果显示，脾和淋巴结的IgA-ASC及IgG-ASC明显升高。结论：成功构建了预防性重组HPV58-减毒志贺氏杆菌载体活疫苗，能诱发免疫动物较强的体液免疫反应，激发保护性抗体的产生。

Construction of prophylactic recombinant HPV58-attenuated Shigella vector live vaccine and evaluation of its protective efficacy and immunogenicity in the Guinea pig Keratoconjunctivitis Model

AIM: To construct the prophylactic recombinant HPV58-attenuated Shigella vector live vaccine and evaluate its protective efficacy and immunogenicity in the Guinea pig Keratoconjunctivitis model. METHODS: The HPV58 L1 gene was cloned into PUCMT, and the recombinant plasmid HPV58 L1-pCVD442 was constructed and introduced into attenuated Shigella (Sf301: deltavirG) with helper plasmid PRK2013 by filter mating. After homologous recombination, the positive colonies in Sf301: deltavirG strains contained HPV58L1 gene were selected and verified by PCR. The expressed HPV58L1 protein was harvested and analyzed by SDS-PAGE and Western blot. The bio-activity of the interested protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was proved with transmission electron microscope. Guinea pig Keratoconjunctivitis model was used to evaluate protective efficacy and immunogenicity of the vaccine. 20 days after immunization, serum HPV58L1-IgG, IgA level and serum sf301 LPS- IgG, IgA level were measured by ELISA, and HPV58 L1-specific IgA-ASC and IgG-ASC of spleen and lymph nodes (SVCLN, MDLN, MSLN and pp) were measured by ELISPOT assay. RESULTS: HPV58 L1 protein with MW 60 kDa was confirmed by Western blot. The ability of the interested protein to self-assemble into VLPs was identified by transmission electron microscope, and the result of murine erythrocyte hemagglutination assay indicated that the given proteins expressed by the recombinant bacillus had similar characteristics as the natural HPV58L1 protein.In the Guinea pig Keratoconjunctivitis Model, animal immune results showed that there were no Keratoconjunctivitis occurred in the immune group(HPV58-attenuated Shigella), and after Sf301 attacking, 80% eyes had no Keratoconjunctivitis occurrence. 20 days after immunization, serum HPV58L1-IgG, IgA level were obviously increased; but serum sf301 LPS-IgG position was just slightly increased; ELISPOT results showed that HPV58 L1-specific IgA-ASC and IgG-ASC of spleen and lymph nodes were also obviously increased. CONCLUSION: Recombinant HPV58L1-attenuated Shigella vector live vaccine could induce strong humoral immune responses in the immunized animals.