结核分枝杆菌Rv2005的原核表达以及免疫原性评估  

Prokaryotic expression and immunogenicity assessment of Mycobacterium tuberculosis Rv2005

作　　者：夏露 王晓春[1] 漆志扬 张延鹏 XIA Lu;WANG Xiaochun;QI Zhiyang;ZHANG Yanpeng(College of Medicine,Anhui University of Science and Technology,Huainan 232001,China;Department of Cosmetology,Collegeof Medicine,Huainan Union University,Huainan 232038,China)

机构地区：[1]安徽理工大学医学院,安徽淮南232001 [2]淮南联合大学医学美容技术系,安徽淮南232038

出　　处：《海南医学院学报》2024年第23期1784-1791,共8页Journal of Hainan Medical University

基　　金：安徽省高校自然科学研究重点项目(KJ2021A1304)。

摘　　要：目的:构建结核分枝杆菌(Mycobacterium tuberculosis,M. tuberculosis)Rv2005载体,进行原核表达以及免疫原性评估研究,为研究新型结核病疫苗筛选优势的靶抗原。方法:设计目的基因Rv2005的特异性引物并进行PCR扩增,依据分子克隆技术构建重组质粒pET28a-Rv2005,对其进行原核表达,并以镍柱亲和层析法对其进行蛋白纯化。用纯化后rRv2005对中国M. tuberculosis感染者和健康对照者(healthy controls, HCs)外周血进行刺激,以多重微球流式免疫荧光发光法检测其外周血T淋巴细胞产生的相应特异性IFN-γ抗体水平。构建基于TLR4/9激动剂单磷酰脂质A/含非甲基化鸟嘌呤和胞嘧啶二核酸的寡聚脱氧核苷酸序列(CPG ODN)的复合型脂质体佐剂MC,与rRv2005联合免疫C57BL/6小鼠,9周后处死,以酶联免疫吸附实验检测免疫后小鼠血清中的特异性抗原IgG,IgG1,IgG2a的滴度以及免疫后小鼠脾细胞上清液中特异性抗原Th1型细胞因子IFN-γ、TNF-α和IL-2的分泌水平。结果:成功构建了重组质粒pET28aRv2005,以SDS-PAGE和Western blot鉴定其表达结果相符合。经rRv2005刺激后,HCs相比于活动性结核病(ATB)与潜伏性结核病(LTBI)受试者,其外周血T淋巴细胞释放的相应细胞因子水平显著升高,且ATB受试者分泌细胞因子水平最高(P<0.01)。卡介苗(BCG)初免、rRv2005联合佐剂MC免疫的小鼠诱导产生较高的IgG及其亚类水平,IgG2a/IgG1比值>1,趋向于Th1型细胞免疫;BCG初免增强组小鼠脾细胞经PPD和rRv2005刺激后,可诱导释放高水平的抗原特异性IFN-γ、TNF-α及IL-2。结论:rRv2005可以诱导中国结核病感染者外周血T淋巴细胞释放高水平的IFN-γ;rRv2005联合佐剂MC免疫小鼠可以刺激机体产生Th1型细胞免疫;BCG初免、rRv2005/MC增强的免疫方式可能有效弥补BCG保护力下降的缺陷;基于TLR激动剂的复合型佐剂MC具有良好的应用前景。Objective:To select advantageous target antigens of vaccine for new tuberculosis(TB)by constructing the Myco‑bacterium(M.tuberculosis)Rv2005 carrier,and assessing prokaryotic expression and immunogenicity.Methods:The specific primers of the target gene Rv2005 were designed and amplified by PCR.The recombinant plasmid pET28a-Rv2005 was construct-ed by molecular cloning technique,expressed prokaryotically,and purified by nickel column affinity chromatography.Peripheral blood of M.tuberculosis infected persons and healthy controls(HCs)was stimulated with purified rRv2005,and the corresponding levels of specific IFN-γantibodies produced by peripheral blood T lymphocytes were detected by multiple microsphere flow immu-nofluorescence assay(MFCIA).A compound liposomal adjuvant MC based on TLR4/9 agonist monophosphoryl lipid A(MPLA)/oligodeoxynucleotide sequence containing non-methylated guanine and cytosine dinucleic acid(CPGODN)was con-structed,which was combined with rRv2005 to immunize C57BL/6 mice,and killed after 9 weeks.Enzyme-linked immunosor-bent assay(ELISA)was used to detect the titers of specific antigens IgG,IgG1,and IgG2a in the serum of immunized mice and the secretion levels of specific antigens Th1 type cytokines IFN-γ,TNF-α,and IL-2 in the supernatant of spleen cells of immu-nized mice.Results:The recombinant plasmid pET28a-Rv2005 was successfully constructed,and its expression was identified by SDS-PAGE and Western blot.After rRv2005 stimulation,compared to active tuberculosis(ATB)and latent tuberculosis infec-tion(LTBI)subjects,the levels of cytokines released by T lymphocytes in peripheral blood of HCs were significantly increased,and the levels of cytokines secreted by ATB subjects were the highest(P<0.01).The mice immunized with BCG and rRv2005 combined with MC produced higher levels of IgG and its subclasses,and the IgG2a/IgG1 ratio was>1,which tended to be Th1 type cell immunity.After PPD and rRv2005 stimulation,the spleen cells in the BCG initial immune enhancement group can induce the rele

关 键 词：结核分枝杆菌 Rv2005 初免-增强策略 佐剂 免疫原性 

分 类 号：R392[医药卫生—免疫学]