淋病奈瑟菌NHBA蛋白经鼻腔免疫后保护效果评估  

Validation of the protective effects of nasal immunisation against NHBA protein of Neisseria gonorrhoeae

作　　者：黄美容 卢琴 杨慧 张宁晴 闵迅 黄健 Huang Meirong;Lu Qin;Yang Hui;Zhang Ningqin;Min Xun;Huang Jian(Department of Blood Transfusion,Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou 563000,China;School of Laboratory Medicine,Zunyi Medical University,Zunyi Guizhou 563006,China;Department of Laboratory Medicine,Hospital of Zunyi Medical University,Zunyi Guizhou 563000,China)

机构地区：[1]遵义医科大学附属医院输血科,贵州遵义563000 [2]遵义医科大学检验医学院,贵州遵义563006 [3]遵义医科大学附属医院医学检验科,贵州遵义563000

出　　处：《遵义医科大学学报》2024年第7期653-658,665,共7页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:82260330)。

摘　　要：目的表达纯化淋病奈瑟菌NHBA蛋白,通过鼻腔免疫途径制备其多克隆抗体并对NHBA抗血清的免疫保护效果进行评估。方法构建NHBA蛋白原核表达菌株并进行目的蛋白的表达及纯化。小鼠随机分为2组:NHBA组:NHBA蛋白+霍乱毒素B亚单位(CTB)佐剂;磷酸盐缓冲液(PBS)组:PBS+CTB佐剂。通过对小鼠进行鼻腔免疫NHBA蛋白制备其多克隆抗体,ELISA测定免疫后血清中IgG抗体效价。蛋白免疫印迹法鉴定NHBA蛋白在淋病奈瑟菌菌株中的表达情况。利用血清杀菌活性实验、调理吞噬杀伤实验及抗体粘附抑制实验评估NHBA抗血清的免疫保护效果。结果成功表达纯化NHBA蛋白。鼻腔免疫小鼠后血清中产生了高滴度的IgG抗体。蛋白印迹实验结果显示抗血清能识别淋病奈瑟菌菌株中天然表达的NHBA蛋白。血清杀菌活性实验和调理吞噬杀伤实验的有效杀菌滴度分别为1/200、1/400。抗体粘附抑制实验结果显示NHBA抗血清能有效抑制淋病奈瑟菌粘附ME-180细胞。结论淋病奈瑟菌NHBA蛋白经鼻腔免疫后获得的抗血清具有免疫保护效果,未来可以作为淋病奈瑟菌鼻黏膜疫苗的有效靶点。Objective To express and purify Neisseria gonorrhoeae NHBA protein,and to prepare polyclonal antibodies against NHBA by the nasal route of immunization and evaluate the immuno-protective effects of NHBA antisera.Methods Constructed prokaryotic expression strains of NHBA protein and carried out the expression and purification of target proteins.Mice were randomized into NHBA group:NHBA protein+cholera toxin B subunit(CTB)adjuvant;phosphate buffer solution(PBS)group:PBS+CTB adjuvant.Polyclonal antibodies against NHBA protein were produced by intranasal immunization of mice and ELISA was used to determine IgG antibody potency in NHBA antisera.NHBA protein expression was verified in Neisseria gonorrhoeae strains by Western Blot.The immune-protective effect of NHBA antisera was assessed using serum bactericidal activity assay,Opsonophagocytic activity assay and antibody adhesion inhibition assay.Results NHBA protein was successfully produced and purified.High titers of IgG antibody were produced in the serum after nasal immunization of mice.Western Blot showed that the antiserum recognized the naturally expressed NHBA protein in the Neisseria gonorrhoeae strain.The effective bactericidal titers in the serum bactericidal assay and the opsonophagocytic activity assay were 1/200 and 1/400,respectively.The result of the antibody adhesion inhibition assay showed that the NHBA antiserum effectively inhibited the adhesion of Neisseria gonorrhoeae to ME-180 cells.Conclusion Neisseria gonorrhoeae NHBA protein immunized nasally to obtain antisera against NHBA,NHBA antisera showed immuno-protective effects,and NHBA could be used as effective targets for Neisseria gonorrhoeae nasal mucosal vaccines in the future.

关 键 词：淋病奈瑟菌 奈瑟菌肝素结合抗原 疫苗 鼻腔免疫 免疫保护 

分 类 号：R759.2[医药卫生—皮肤病学与性病学]