结核分枝杆菌ESAT6-CFP10融合蛋白诱导的小鼠免疫应答及其保护力

目的:研究结核分枝杆菌(MTB)ESAT6-CFP10融合蛋白诱导的小鼠体液和细胞免疫应答以及对MTB感染小鼠的保护能力。方法:用预先转移到硝酸纤维素膜条的ES-AT6-CFP10融合蛋白采用皮下包埋的方法免疫小鼠。用MTB培养上清滤液蛋白(CFP)作为抗原,用ELISA法测定免疫小鼠血清特异性抗体的滴度。末次免疫后,取部分免疫小鼠脾淋巴细胞,体外用ESAT6-CFP10融合蛋白刺激后,以MTT比色法检测淋巴细胞增殖反应,同时检测免疫小鼠IFN-γ和IL-2水平及脾细胞杀伤效应。另一部分免疫的BALB/c小鼠经尾静脉感染MTB毒株H37Rv,4周后计数脾脏中的细菌数。结果:ESAT6-CFP10融合蛋白免疫小鼠血清特异性抗体的滴度为1∶6400。淋巴细胞刺激增殖指数为1.90±0.15,明显高于生理盐水组(0.90±0.15);IFN-γ和IL-2的含量分别为1.792±19ng/L和0.211±11ng/L,显著高于生理盐水对照组,但不及BCG免疫组;同时融合蛋白诱导的脾细胞杀伤率为36%。与生理盐水免疫组(细菌负荷6.51±0.13)相比较,融和蛋白免疫的BALB/c小鼠,对攻击感染后MTB在脾脏中增殖有显著作用(细菌负荷为5.24±0.15,P<0.05),但与BCG免疫组相比脾脏中细菌负荷减少甚微。结论:ESAT6-CFP10融合蛋白可作为新型结核疫苗的候选组分。     

结核分枝杆菌Rv3245 蛋白抗原免疫学特性的研究

目的:研究结核分枝杆菌Rv3425 蛋白免疫学特性,评估其诊断应用价值及在结核致病性中的作用。方法:将Rv3425 基因克隆至pET28a 载体中,诱导表达Rv3425 融合蛋白并进行纯化;利用ELISA、Western blot 分析其抗原性与特异性;流式检测该抗原对巨噬细胞凋亡的影响。结果:获得了可溶性原核表达融合蛋白Rv3425,Rv3425 蛋白能刺激灭活结核分枝杆菌免疫小鼠脾细胞产生高水平的特异性IFN鄄酌、纯化的Rv3425 蛋白能与结核感染小鼠血清特异性结合,其特异性血清抗体IgG 及IgM 在结核病人中的水平明显高于健康人,发现该蛋白能诱导巨噬细胞的凋亡。结论:本研究发现结核分枝杆菌Rv3425 蛋白具有较强的抗原性且能促进巨噬细胞的凋亡,这些发现对于结核病的诊断及致病机制的研究具有重要价值。     

结核分枝杆菌Rv0901基因重组耻垢分枝杆菌的构建及其细胞作用

目的对结核分枝杆菌Rv0901基因的功能进行研究。方法以PCR扩增Rv0901基因编码序列,定向克隆人穿梭表达质粒pMV261获得重组穿梭表达质粒,将重组质粒电穿孔进入耻垢分枝杆菌,构建重组Rv0901基因的耻垢分枝杆菌,对重组耻垢分枝杆菌进行诱导表达,用SDS-PAGE检测表达结果。比较耻垢分枝杆菌和重组耻垢分枝杆菌对THP-1细胞的不同作用。结果成功构建重组穿梭表达质粒及重组Rv0901基因的耻垢分枝杆菌,重组菌诱导THP-1细胞的凋亡率高于耻垢分枝杆菌,其感染THP-1细胞后细胞的存活率低于耻垢分枝杆菌的感染,重组菌感染THP-1细胞后细胞培养液中NO(一氧化氮)的产生高于耻垢分枝杆菌的感染。结论Rv0901基因可能与结核毒力存在一定关系。     

结核分枝杆菌Rv0073基因在大肠杆菌中的表达及纯化

目的 构建结核分枝杆菌(MTB) Rv0073基因原核表达载体并进行表达和纯化.方法 以MTB H37Rv基因组DNA为模板,采用聚合酶链反应(PCR)扩增目的基因片段,构建原核表达载体pET26b-Rv0073,经测序确定无误后转化至大肠杆菌(E.coli)感受态细胞BL21中.用聚丙烯酰氨凝胶电泳(SDS-PAGE)方法检测重组蛋白表达,检测异丙基-β-D-硫代半乳糖苷(IPTG)诱导不同时间、不同温度条件下重组蛋白表达量.采用His镍磁珠进行外源蛋白小量纯化.结果 成功构建重组表达质粒,重组蛋白经IPTG诱导后,2h开始明显表达且表达量无时间依赖性,在不同温度诱导下,重组蛋白的表达量随温度的增高而减少.重组蛋白以包涵体形式存在,经His镍磁珠纯化后获得重组蛋白.结论 成功构建并表达Rv0073蛋白,为后续Rv0073的大量纯化及其功能研究奠定了基础.     

结核分枝杆菌抗原Rv0446c人T细胞抗原表位鉴定及其免疫特征的分析

目的:研究结核分枝杆菌Rv0446c的抗原表位及其免疫原性,为结核病的免疫诊断技术及疫苗研发提供候选抗原及表位。方法:利用生物信息学软件TE-predict和IEDB的T细胞表位预测软件对结核分枝杆菌抗原Rv0446c进行T细胞表位预测,用ELISPOT实验检测表位在2019年1月到2020年12月期间来自佛山市第四人...     

结核分枝杆菌Esx-1 底物蛋白Rv3615c 的原核表达及其免疫功能研究

目的:构建结核分枝杆菌(M.tb)Esx-1 底物蛋白Rv3615c 的原核表达质粒pET30b-Rv3615c 并在大肠杆菌BL21(DE3)中进行原核表达;以全血IFN-β分析试验(WBIA)检测rRv3615c 蛋白能否被淮南市M.tb 感染者T 细胞所识别;在小鼠模型中检测其免疫原性。方法:以分子克隆技术构建重组载体pET30b-Rv3615c 并表达、纯化rRv3615c 蛋白,以WBIA 检测rRv3615c 抗原刺激淮南市M.tb 感染者和未感染者外周血淋巴细胞产生的特异性IFN-β水平。此外联合佐剂SAS 免疫C57BL/6 小鼠,评价其体液免疫应答和Th1 型应答水平。结果:成功构建pET30b-Rv3615c 质粒,SDS-PAGE 和Western blot 显示其正确表达和纯化。rRv3615c 蛋白特异性刺激淮南市M.tb 感染者淋巴细胞产生的IFN-β浓度显著高于健康对照者。Rv3615c/ SAS 诱导小鼠产生的IgG、IgG1、IgG2a 以及Rv3615c1SAS 诱导小鼠脾淋巴细胞分泌的特异性IFN-β、TNF-β和IL-2,均显著高于PBS 组和SAS 组,显示rRv3615c 可诱导趋向于Th1 型免疫应答。结论:Rv3615c 可被淮南市M.tb 感染者T 细胞识别,具有较强的免疫原性,是用于结核病(TB)预防和诊断的潜力靶抗原。     

结核分枝杆菌毒力分泌基因Rv3872重组卡介苗的构建及表达

目的:构建结核分枝杆菌Rv3872的原核重组表达质粒pET-3872并对其进行表达及鉴定。构建表达结核分枝杆菌抗原蛋白Rv3872即PE35的重组卡介苗。方法:应用PCR技术扩增Rv3872基因,定向克隆入pET32a(+)并转化E.coli BL21(DE3)菌株,测序后用IPTG诱导蛋白表达,通过SDS-PAGE和Western blot对目的蛋白进行检测及鉴定。用纯化蛋白免疫新西兰大白兔制备多克隆抗体。构建重组穿梭表达质粒pMV-3872,将重组质粒电穿孔进入卡介苗,对重组卡介苗进行诱导表达用SDS-PAGE和Western blot检测和鉴定目的蛋白。结果:表达融合蛋白的pET-3872质粒构建成功,重组蛋白经Western blot检测出特异性阳性信号。重组卡介苗BCG-3872构建成功,热诱导表达后经SDS-PAGE和Western blot,在培养上清中检测到目的蛋白。结论:成功构建了重组质粒pET32a(+),并在大肠杆菌中表达了PE35蛋白,有利于进一步研究Rv3872基因功能。本研究还对表达结核分枝杆菌蛋白PE35的重组卡介苗进行了鉴定,为进一步研究该重组卡介苗的免疫功能奠定了基础。     

用DNAStar软件预测Rv1410c结核分枝杆菌蛋白抗原表位

目的预测Rv1410c结核分枝杆菌(MTB)的抗原表位。方法利用DNAStar软件包中Editseq软件将Rv1410c MTB氨基酸序列进行编辑保存,然后利用Protean软件进行氨基酸序列分析,预测Rv1410c MTB的二级结构、B细胞抗原表位及T细胞抗原表位,然后再用BLAST分析其与人类抗原表位的同源性。结果 Rv1410c具有丰富的二级结构,含有较多潜在的B细胞抗原肽表位,主要位于1~10、67~77、129~137、189~205、222~235、253~267、296~306、330~338、359~367、462~467、494~517氨基酸残基或其附近,这些区域基本上含有β转角结构,亲水性、表面可能性和柔韧性指数都较高;也含有较多潜在的T细胞表位,主要位于16~37、84~102、108~115、137~160、202~207、219~222、245~263、321~325、355~362、387~391、417~445、486~494氨基酸残基或其附近。结论 Rv1410c MTB既含有较多潜在的B细胞抗原表位,也含有较多潜在的T细胞抗原表位。     

采用基因打靶技术构建结核分枝杆菌新基因Rv0901缺失株的研究

目的：利用基因打靶技术构建基因打靶载体和基因缺失株，用于结核分枝杆菌基因Rv0901功能的研究。方法：结核分枝杆菌标准株H37Rv体外培养，对其Rv0901基因及两侧序列进行体外扩增，连接载体及目的片段，切除目的基因，再引入筛选标志构建重组自杀质粒，分别用酶切及PCR鉴定；用电穿孔法将重组自杀质粒转入结核杆菌H37Rv株，用PCR鉴定。结果：经鉴定PCR产物及插入片段大小与预期值相符，且为所需目的基因片段，成功切除靶片段；蓝白斑筛选证实标记基因插入片段插入方向正确；Rv0901基因缺失株用PCR鉴定成功缺失了目的基因片段2.5 kb。结论：成功构建了用于结核分枝杆菌基因打靶的置换型载体和Rv0901新基因缺失株，为Rv0901基因功能的研究奠定了基础。     

结核分枝杆菌Rv3881c抗原鼠单克隆抗体的制备及初步鉴定

目的:制备抗结核分枝杆菌Rv3881c抗原鼠mAb。方法:采用杂交瘤技术,获得了11株针对结核分枝杆菌Rv3881c抗原鼠mAb杂交瘤细胞株,对其中的5株进行了小鼠腹水的制备及相关鉴定。结果:5株mAb的腹水效价达到1∶32 000～1∶512 000,将这5株mAb进行了纯化,纯化后纯度大于90%,抗体亚类(型)均为IgG1/κ型,ELISA结果显示制备的mAb与结核分枝杆菌Rv3881c抗原可发生特异反应。结论:制备了抗结核分枝杆菌Rv3881c抗原鼠mAb,为结核分枝杆菌Rv3881c生物学功能的研究奠定基础。     

结核分枝杆菌Rv1626的原核表达及其免疫功能研究

结核分枝杆菌;Rv1626;原核表达;免疫原性;结核病     

The DosR antigen Rv1737c from Mycobacterium tuberculosis confers inflammation regulation in tuberculosis infection

There is an urgent need to identify the potential risk factors for activating latent Mycobacterium tuberculosis infection. In this study, we evaluated the immune function of Rv1737c, which is a latency‐associated antigen of dormancy survival regulator (DosR) of M. tuberculosis in a mouse model. Our data showed that mice pretreated with recombinant Rv1737c (rRv1737c) exhibited higher levels of antigen‐specific antibodies (IgG, IgM and IgA) than sham‐treated mice. Following Bacilli Calmette‐Guerin (BCG) challenge, rRv1737c adjuvanted with cholera toxin subunit B (CTB) induced diffuse lung inflammation and fibrosis compared to the control mice. The inflammatory pathogenesis due to rRv1737c pre‐exposure was associated with a switch in the macrophage phenotype from M1 to activated M2 and was characterized by IL‐10 production. Intracellular cytokine analysis further showed that the rRv1737c‐pretreated mice exhibited an increased frequency of Th2 cells in the lungs, lymph nodes and spleen after BCG challenge. Furthermore, IFN‐γ expression increased in the lungs after rRv1737c pretreatment compared to that in the sham mice. Accordingly, lung cells from rRv1737c‐immunized mice stimulated with killed BCG produced higher levels of multiple cytokines, such as IFN‐γ, IL‐10 and IL‐6. The results confirmed that the pathological features of rRv1737c promoted inflammation. Overall, our findings provide direct evidence of the pro‐inflammatory function of rRv1737c in a murine model of BCG infection, indicating that Rv1737c is a pathogenic antigen of M. tuberculosis and may be key to the recurrence of latent infection.     

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a potential B-cell antigen used for serological diagnosis to distinguish vaccinated controls from tuberculosis patients

Proteins encoded by a 9.5-kb DNA segment, termed the region of difference (RD), of Mycobacterium tuberculosis have been demonstrated to be important in bacterial virulence, vaccine development and the design of diagnostic reagents. This study evaluated the immunogenic properties of Rv3425, a member of the PPE family of proteins, encoded by an open reading frame found in RD11 of M. tuberculosis, in comparison with two other well-known antigens, the early secreted antigen target 6 (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). RT-PCR demonstrated that Rv3425 mRNA is expressed in liquid culture by M. tuberculosis H37Rv. When tested in a conventional ELISA in the form of a His-tagged recombinant protein, Rv3425 revealed a statistically significant antigenic distinction between healthy bacille Calmette-Guérin (BCG)-vaccinated controls and tuberculosis (TB) patients (p <0.0001). The anti-IgG response to recombinant Rv3425 was almost equal to that for CFP-10, and was higher than that for ESAT-6. The results highlight the immunosensitive and immunospecific nature of Rv3425, which shows promise for use in the serodiagnosis of TB.     

Robust immune response elicited by a novel and unique Mycobacterium tuberculosis protein using an optimized DNA/protein heterologous prime/boost protocol

An efficacious tuberculosis (TB) vaccine will probably need to induce both CD4 and CD8 T‐cell responses specific to a protective Mycobacterium tuberculosis antigen(s). To achieve this broad cellular immune response we tested a heterologous DNA/protein combination vaccine strategy. We used a purified recombinant protein preparation of a unique M. tuberculosis antigen (rMT1721) found in the urine of TB patients, an optimized plasmid DNA expressing this protein (DNA‐MT1721), and a Toll‐like receptor 4 agonist adjuvant. We found that priming mice with DNA‐MT1721 and subsequently boosting with rMT1721 elicited high titres of specific IgG1 and IgG2a antibodies as well as high magnitude and polyfunctional CD4⁺ T‐cell responses. However, no detectable CD8⁺ T‐cell response was observed using this regimen of immunization. In contrast, both CD4⁺ and CD8⁺ T‐cell responses were detected after a prime/boost vaccination regimen using rMT1721 as the priming antigen and DNA‐MT1721 as the boosting immunogen. These findings support the exploration of heterologous DNA/protein immunization strategies in vaccine development against TB and possibly other infectious diseases.     

Mycobacterium tuberculosis Rv0652 stimulates production of tumour necrosis factor and monocytes chemoattractant protein-1 in macrophages through the Toll-like receptor 4 pathway

Mycobacterial proteins interact with host macrophages and modulate their functions and cytokine gene expression profile. The protein Rv0652 is abundant in culture filtrates of Mycobacterium tuberculosis K‐strain, which belongs to the Beijing family, compared with levels in the H37Rv and CDC1551 strains. Rv0652 induces strong antibody responses in patients with active tuberculosis. We investigated pro‐inflammatory cytokine production induced by Rv0652 in murine macrophages and the roles of signalling pathways. In RAW264.7 cells and bone marrow‐derived macrophages, recombinant Rv0652 induced predominantly tumour necrosis factor (TNF) and monocyte chemoattractant protein (MCP)‐1 production, which was dependent on mitogen‐activated protein kinases and nuclear factor‐κB. Specific signalling pathway inhibitors revealed that the extracellular signal‐regulated kinase 1/2 (ERK1/2), p38 and phosphatidylinositol 3‐kinase (PI3K) pathways were essential for Rv0652‐induced TNF production, whereas the ERK1/2 and PI3K pathways, but not the p38 pathway, were critical for MCP‐1 production in macrophages. Rv0652‐stimulated TNF and MCP‐1 secretion by macrophages occurred in a Toll‐like receptor 4‐dependent and MyD88‐dependent manner. In addition, Rv0652 significantly up‐regulated the expression of the mannose receptor, CD80, CD86 and MHC class II molecules. These results suggest that Rv0652 can induce a protective immunity against M. tuberculosis through the macrophage activation.     

结核分支杆菌Rv0901基因置换型打靶载体的构建

目的：对结核杆菌H37Rv的Rv0901基因进行体外扩增，构建基因打靶载体，利用基因打靶技术进行结核分支杆菌基因Rv0901功能的研究。方法：结核分支杆菌标准毒力株H37Rv体外培界，扩增目的基因，连接载体及目的片段，切除目的基因，筛选阳性克隆，酶切鉴定。结果：经酶切鉴定PCR产物及插入片段大小与预期值相符，鉴定证实PCR产物及插入片段为所需目的基因片段，成功切除靶片段。证实标记基因插入片段插入方向正确。结论：成功构建了用于结核分支杆菌基因打靶的置换型载体，为随后将进行的Rv0901基因敲除株的建立，Rv0901基因功能的研究奠定了基础。     

Mcl-1信号通路阻断剂对H37Rv感染小鼠巨噬细胞凋亡的影响

目的:探讨Mcl-1信号通路阻断剂在结核分枝杆菌H37Rv感染小鼠模型中对Mcl-1表达、巨噬细胞凋亡情况及结核分枝杆菌的影响。方法:小鼠腹腔注射H37Rv菌悬液,建立感染小鼠模型,针对Mcl-1的信号通路选用JAK/STAT信号通路阻断剂AG490、MAPK信号通路阻断剂PD98059和PI3K信号通路阻断剂LY294002用腹腔注射方式作用于各组感染小鼠模型,分为H37Rv感染组、AG490处理组、PD98059处理组、LY294002处理组和对照组。通过细胞抗酸染色观察结核分枝杆菌H37Rv感染小鼠腹腔巨噬细胞的动物模型是否建立成功;通过免疫细胞化学检测结核分枝杆菌H37Rv感染巨噬细胞的Mcl-1表达情况,使用流式细胞技术检测各组巨噬细胞的凋亡率,采用结核分枝杆菌菌落计数来判断巨噬细胞凋亡对结核分枝杆菌的清除效果。结果:细胞抗酸染色结果可见感染的巨噬细胞内散在排列的红色短小抗酸结核分枝杆菌。免疫细胞化学结果显示H37Rv感染组、AG490处理组和LY294002处理组中的Mcl-1蛋白为强阳性表达,PD98059处理组中Mcl-1蛋白为弱阳性表达,对照组Mcl-1蛋白为阴性表达。流式细胞术检测发现H37Rv感染组巨噬细胞凋亡率较对照组高,PD98059处理组的凋亡率显著高于各组,差异显著(P0.05)。结核分枝杆菌菌落计数结果显示PD98059处理组对H37Rv菌株抑菌作用最明显。结论:Mcl-1信号通路阻断剂通过抑制JAK/STAT、MAPK和PI3K信号通路增加结核分枝杆菌H37Rv感染巨噬细胞的凋亡率,抑制结核分枝杆菌生长;其中,MAPK信号通路干扰Mcl-1的作用最明显,感染的巨噬细胞凋亡率最高,抑菌作用最强。     

二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究

目的 探讨二甲基三十六烷基铵(dimo-thylidioctyl ammonium bromide,DDA)和卡介苗多糖核酸(BCC-PSN)佐剂在结核融合蛋白疫苗加强卡介苗(BCG)免疫中的不同免疫辅助效应.方法 选择DDA、BCG-PSN作为融合蛋白AMM(Ag85B-MPT64190-198-Mtb8.4)的佐剂,在BCG初免小鼠后,AMM疫苗加强免疫两次,其中一组联合使用两种佐剂(DDA/BCG-PSN),另一组单独以DDA作为佐剂,同时设立BCG或磷酸缓冲液(PBS)免疫组为对照.应用ELISA及ELISPOT检测免疫小鼠的体液与细胞免疫反应,最后一次加强免疫后第12周,以H37Rv尾静脉攻毒并检测小鼠肺脾组织细菌载量和病理改变,评价不同佐剂疫苗的保护效果.结果 在BCG初免基础上,联合佐剂组(AMM/DDA/BCG-PSN)和单独佐剂组(AMM/DDA)加强免疫两次后,脾脏淋巴细胞经抗原Ag85B和PPD(purified protein derivative)刺激后,皆可产生分泌较BCG组高的IFN-γ.毒力株攻击后菌落形成单位(colony-forming unit,CFU)计数显示,联合佐剂组(AMM/DDA/BCG-PSN)脾脏荷菌量少于PBS组和BCG组(P<0.05);而单独佐剂组(AMM/DDA)肺部荷菌量少于PBS组和BCG组(P<0.05).组织病理分析结果 表明AMM/DDA/BCG-PSN组肺组织病理损伤较轻,而AMM/DDA组病理损伤个体差异较大.结论 DDA是较为理想的结核亚单位疫苗佐剂,能诱导较强的细胞免疫和免疫保护作用;BCG-PSN可能具有免疫调节作用,可以减轻免疫病理损伤.