摘要
目的构建表达结核分枝杆菌(M.tb)c-di-AMP磷酸二酯酶CnpB的重组腺病毒(rAd)并检测其诱导的体液免疫应答。方法经密码子优化的M.tb CnpB编码基因克隆入质粒pcADV,获得的重组质粒pcADV-CnpB转染HEK293T细胞,并用Western blot鉴定表达。重组pcADV-CnpB与骨架质粒共转染HEK293T细胞包装获得重组腺病毒rAd-CnpB,以HEK293T细胞扩增rAd-CnpB,Western blot和免疫荧光检测rAd-CnpB表达目的蛋白。以rAd-CnpB滴鼻免疫小鼠,收集小鼠血清与肺泡灌洗液(BALF),ELISA检测抗原特异性抗体水平。结果酶切和测序鉴定表明重组质粒pcADV-CnpB构建成功,该质粒能够在真核细胞中表达。在HEK293T细胞内包装并获得rAd-CnpB,大量扩增并纯化获得滴度为5.53×10^(10) PFU/mL的rAd-CnpB。rAd-CnpB可在HEK293T细胞中表达目的蛋白CnpB。rAd-CnpB滴鼻免疫小鼠,可诱导BALF中CnpB特异性IgG与分泌型IgA水平的增加,同时诱导血清中产生高水平的CnpB特异性IgG。结论成功构建表达M.tb c-di-AMP磷酸二酯酶CnpB的重组腺病毒rAd-CnpB,并且rAd-CnpB经黏膜免疫可诱导抗原特异性体液免疫与黏膜免疫应答,可用于结核病新疫苗的研究。
A recombinant adenovirus(rAd)for expression of Mycobacterium tuberculosis(M.tb)c-di-AMP phosphodiesterase CnpB was constructed,and its induced humoral immune response was detected.The codon-optimized gene of M.tb CnpB was cloned into the adenoviral plasmid pcADV.The recombinant plasmid pcADV-CnpB was transfected into HEK293T cells,and expression was detected with Western blot.The recombinant plasmid pcADV-CnpB and the backbone plasmid were co-transfected into HEK293T cells to obtain the recombinant adenovirus rAd-CnpB.rAd-CnpB was amplified in HEK293T cells,and the target protein expression of rAd-CnpB was detected with Western blot and immunofluorescence.Mice were immunized with rAd-CnpB intranasally,and their sera and bronchoalveolar lavage fluid(BALF)were collected.ELISA was used to detect levels of antigen-specific antibodies.Restriction enzyme digestion and sequencing indicated that the recombinant plasmid pcADV-CnpB was successfully constructed and led to protein expression in eukaryotic cells.rAd-CnpB was packaged and produced in HEK293T cells.After amplification and purification,rAd-CnpB with a titer of 5.53×10^(10) PFU/mL was obtained.rAd-CnpB led to CnpB expression in HEK293T cells.Intranasal immunization with rAd-CnpB increased levels of IgG and secretory IgA in BALF and led to high levels of IgG in sera.rAd-CnpB,the recombinant adenovirus for expression of c-di-AMP phosphodiesterase CnpB was successfully constructed,and was found to induce antigen-specific humoral and mucosal immune responses through mucosal immunization.Thus,rAd-CnpB may be used in further research on new TB vaccine strategies.
作者
胡家豪
宁唤唤
董梦娟
路延之
代婷
张从玥
徐子晴
王姝予
周正言
柏银兰
HU Jia-hao;NING Huan-huan;DONG Meng-juan;LU Yan-zhi;DAI Ting;ZHANG Cong-yue;XU Zi-qing;WANG Shu-yu;ZHOU Zheng-yan;BAI Yin-lan(Department of Microbiology and Pathogen Biology,School of Basic Medicine,Air Force Medical University,Xi’an 710032,China;Cadet Regiment,School of Basic Medicine,Air Force Medical University,Xi’an 710032,China;Military Medical Innovation Center,Air Force Medical University,Xi’an 710032,China;Department of Physiology and Neurobiology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750001,China;College of Life Sciences,Yan’an University,Yan’an 716000,China)
出处
《中国人兽共患病学报》
北大核心
2025年第4期364-369,共6页
Chinese Journal of Zoonoses
基金
国家自然科学基金项目(No.82272343,No.81971560)
陕西省重点研发计划(No.2022ZDLSF01-07)
空军军医大学本科生导师项目(No.2023020)。
作者简介
通讯作者:柏银兰,Email:yinlanbai@fmmu.edu.cn,ORCID:0000-0001-5867-5308;通讯作者:宁唤唤,Email:ninghuanhuan2014@163.com,ORCID:0000-0002-8179-4716。
