Rv1813c在结核分枝杆菌潜伏感染诊断中的价值

目的 研究重组结核分枝杆菌潜伏感染蛋白Rv1813c在诊断结核分枝杆菌潜伏感染方面的价值.方法 20例结核潜伏感染者和79例健康志愿者均行胸部X线检查、PPD皮肤试验、结核抗体检测,同时应用ELISA联合重组融合蛋白CFP10-ESAT6和潜伏感染蛋白Rv1813c进行IGRA检测.结果 所有受试者中,PPD皮肤试验和抗体检测的阳性率分别为50％和28％.Rv1813c刺激潜伏感染者后产生IFN-γ的水平显著高于健康对照(P＜0.05),其刺激PPD强阳性组(皮试直径≥15mm)产生的IFN-γ值低于弱阳性组(5mm≤皮试直径＜15mm),但无统计学意义,而CFP10-ESAT6刺激PPD强阳性组后产生的IFN-γ值高于弱阳性组(P＜0.05).CFP10-ESAT6和Rv1813c刺激抗体阴性及阳性组后产生的IFN-γ水平没有显著性差异(P＞0.05).Rv1813c诊断结核感染的ROC曲线下面积为0.834,特异性80％时,灵敏度为85％;特异性为90％时,灵敏度为45％.结论 同时检测Rv1813c及CFP10-ESAT6抗原特异的IFN-γ值有可能筛选出潜伏感染者中的活动感染人群,对于结核分枝杆菌潜伏感染诊断有重要的应用价值.     

小鼠巨噬细胞内表达结核分枝杆菌CFP10-ESAT6融合蛋白对细胞增殖和凋亡的影响

 目的：建立培养滤液蛋白10-早期分泌性抗原靶6（CFP10-ESAT6）真核表达质粒并转染RAW2647巨噬细胞,观察胞内表达CFP10-ESAT6对细胞活性及凋亡的影响,并初步探讨其机制。方法：将CFP10-ESAT6融合基因插入真核表达质粒pEGFP-N1,构建重组质粒,转染RAW264.7巨噬细胞。采用MTT的方法测定CFP10-ESAT6融合蛋白对RAW264.7巨噬细胞活性的影响,采用结核分枝杆菌19 kD脂蛋白和十字孢碱（staurosporine）处理RAW264.7巨噬细胞,并以流式细胞术检测细胞凋亡率以及Toll样受体2（TLR2）的表达。结果：成功构建了重组质粒pEGFP-N1/CFP10-ESAT6并转染至RAW264.7巨噬细胞;与对照组相比,胞内表达CFP10-ESAT6不能影响巨噬细胞的活性,但可以明显抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡(P＜0.05),并显著抑制了TLR2的表达(P＜0.05)。结论：巨噬细胞内表达的CFP10-ESAT6融合蛋白不具有细胞毒性作用,但可以通过下调TLR2的表达来抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡。     

结核分枝杆菌重组蛋白CFP-10-ESAT-6-PPE68的表达、纯化与多克隆抗体的制备

目的构建重组质粒pET32a（＋）-cfp-10-esat-6-ppe68,得到重组结核分枝杆菌CFP-10-ESAT-6-PPE68蛋白及兔抗结核分枝杆菌CFP-10-ESAT-6-PPE68蛋白的多克隆抗体,为下-步应用于临床结核病的检测奠定基础。方法将结核分枝杆菌cfp-10-esat-6-ppe68基因转入质粒pET32a,将经PCR、酶切、序列测定鉴定为阳性的质粒转人大肠杆菌DE3,IPTG诱导表达重组蛋白,经亲和层析法纯化重组蛋白后用凝血酶切除载体蛋白,免疫家兔制备多克隆抗体并进行纯化。结果成功构建重组质粒表达体系pET32a（＋）-cfp-10-esat-6-ppe68,制得去除载体蛋白的高纯度重组蛋白CFP-10．ESAT-6-PPE68,并成功得到高纯度抗结核分枝杆菌CFP-10-ESAT-6。PPE68蛋白的抗体。结论成功表达结核重组蛋白并制得高纯度多克隆抗体。     

早期诊断结核性脑膜炎的新方法:检测浓缩脑脊液中CFP-10和ESAT-6

目的:应用酶联免疫吸附试验检测浓缩的脑脊液中结核分枝杆菌特异性抗原培养滤液蛋白10(CFP10)和6000早期分泌性抗原靶(ESAT-6),评价其在结核性脑膜炎(TBM)早期诊断中的价值。方法:应用ELISA测定46例临床诊断为TBM和56例非TBM患者脑脊液原液及经透析袋浓缩10倍的脑脊液浓缩液中CFP10和ESAT-6。结果:TBM患者脑脊液原液CFP10检测敏感度为13.04%、特异度为100.00%,ESAT-6的敏感度为13.04%、特异度为100.00%;而浓缩液CFP10的敏感度为78.26%、特异度为96.42%,ESAT-6的敏感度为76.09%、特异度为98.18%。结论:应用反透析方法浓缩脑脊液,使用抗CFP10和ESAT-6 ELISA检测脑脊液CFP10和ESAT-6能显著提高其敏感度,是一种简单、快速早期诊断结核性脑膜炎的有效辅助方法。     

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。     

新型布尼亚病毒抗体间接ELISA检测方法的建立北大核心CSCD

目的本研究旨在建立新型布尼亚病毒抗体检测的高特异性、低成本间接ELISA方法。方法通过优化抗原表达、纯化、包被、血清稀释、孵育时间等条件,以Gn的截短蛋白Gn2为包被抗原,开发了一种间接ELISA方法。检测该方法的稳定性、敏感性、特异性、符合率。结果批内和批间变异系数均小于10%,阳性血清临床符合率达76.9%(10/13),阴性血清临床符合率达86.7%(26/30)。结论本研究所建立的间接ELISA方法可用于临床血清样品的检测,为发热伴血小板减少综合征的疾病诊断和监测提供了较为可靠和廉价的检测方法。     

结核分枝杆菌PPE68蛋白的表达、鉴定及抗血清的制备

目的在大肠杆菌中表达结核分枝杆菌PPE68蛋白,鉴定并纯化重组rPPE68蛋白后免疫新西兰大白兔,制备兔抗PPE68多克隆抗体。方法将已经过鉴定的重组原核表达质粒pET32a(+)-PPE68转化至大肠杆菌BL21中,IPTG诱导大量表达PPE68蛋白。对表达蛋白进行鉴定后利用亲和层析法进行纯化。以纯化后重组rPPE68为免疫抗原,与弗氏不完全佐剂等体积混合,采用皮下多点注射法免疫新西兰大白兔,于第3次免疫后的第7天采血。用凝集实验与Western-blot检测抗血清的特异性,并用双向免疫扩散法测定抗血清效价。结果在大肠杆菌中表达的目的产物经SDS-PAGE分析,在相对分子质量57 000处可见特异性条带,免疫印迹分析证实表达的目的蛋白可与结核分枝杆菌H37Rv株感染小鼠的血清发生反应。纯化的rPPE68蛋白免疫新西兰大白兔后,凝集反应与Western-blot证实了抗血清的特异性,双向免疫扩散法测得血清效价为1∶16。结论已成功表达结核分枝杆菌PPE68蛋白,制备了特异性兔抗PPE68多克隆抗体,对结核病的早期诊断提供了一种新的思路。     

结核、血吸虫抗体快速联检方法的建立与应用

目的创建一种新的结核、血吸虫抗体快速联合检测方法,并评价其在疾病预防中的潜在应用价值。方法以血吸虫虫卵可溶性抗原（SEA）和结核分枝杆菌脂阿拉伯甘露糖（LAM）作为检测用抗原,分别点样在硝酸纤维素膜上,以胶体金-蛋白A结合物为检测标记物,采用自行设计的垂直流检测装置为抗原抗体反应体系．建立结核抗体、血吸虫抗体快速联合检测的金标免疫渗滤法（TB／Sj—DIGFA）;用TB／Sj—DIGFA检测肺结核病人、血吸虫病人、健康人及流动人口血清样本,以敏感性、特异性等指标考核其诊断价值及应用价值。结果TB／Sj—DIGFA检测36份肺结核病人血清和40份血吸虫病人血清的敏感性分别为94．4％（34／36）和100％（40／40）;检测50人份健康人血清,对结核病和血吸虫病的特异性分别为80．0％（40／50）和100％（50／50）。应用新创建的TB／Sj-DIGFA方法调查3120流动人口,查出结核抗体阳性675份,阳性率为21．6％（675／3120）,血吸虫抗体阳性38人份．阳性率为1．22％（38／3120）。结论TB／Sj—DIGFA检测结核和血吸虫抗体具有较高的敏感性和特异性。该方法一次操作可同时完成结核和血吸虫2项特异性抗体测定,检测效率高,可节省大量人力和物力。     

酶联捕获法检测抗人巨细胞病毒-IgM抗体的实验分析

目的 建立检测人巨细胞病毒(HCMV)抗原特异性IgM抗体的酶联捕获技术。方法 应用酶联捕获法检测68例巨细胞病毒感染者血清中抗HCMV-IgM，并与常用的间接ELISA法进行比较。结果 酶联捕获法特异性及敏感性均高于间接ELISA法，且不受RF因子的影响。结论 酶联捕获法敏感性高、特异性强、简便、快速，重复性好，具有实际应用价值。     

ELISA检测胸水结核抗体的初步研究

ELISA检测血清结核抗体的研究国内外已有不少报道,但尚未见用于胸水结核抗体的检测。本文以PPD为抗原,用ELISA间接法检测了41例结核性胸膜炎、40例其它疾病患者胸水及血清中的特异性IgG抗体,以探讨其在结核性胸膜炎中的诊断价值。     

Optimizing antigen cocktails for detection of Mycobacterium bovis in herds with different prevalences of bovine tuberculosis: ESAT6-CFP10 mixture shows optimal sensitivity and specificity

Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic cocktail.     

Recombinant adenovirus delivery of calreticulin-ESAT-6 produces an antigen-specific immune response but no protection against a Mycobacterium tuberculosis challenge

Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT‐6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell‐mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT‐6 to calreticulin and constructed a recombinant replication‐deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT‐6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT‐6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon‐γ and tumour necrosis factor‐α in response to ESAT‐6. This immune response was not improved by the addition of CFP‐10 to the CRT‐ESAT‐6 fusion protein (AdCRT–ESAT‐6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low‐dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.     

Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10

Tests based on tuberculin purified protein derivative (PPD) cannot distinguish between tuberculosis infection, Mycobacterium bovis BCG vaccination, or exposure to environmental mycobacteria. The present study investigated the diagnostic potential of two Mycobacterium tuberculosis-specific antigens (ESAT-6 and CFP10) in experimental animals as well as during natural infection in humans and cattle. Both antigens were frequently recognized in vivo and in vitro based on the induction of delayed-type hypersensitivity responses and the ability to induce gamma interferon production by lymphocytes, respectively. The combination of ESAT-6 and CFP10 was found to be highly sensitive and specific for both in vivo and in vitro diagnosis. In humans, the combination had a high sensitivity (73%) and a much higher specificity (93%) than PPD (7%).     

Efficient testing of large pools of Mycobacterium tuberculosis RD1 peptides and identification of major antigens and immunodominant peptides recognized by human Th1 cells

Comparative genomics has identified several regions of difference (RDs) of Mycobacterium tuberculosis that are deleted or absent in Mycobacterium bovis BCG vaccines. To determine their relevance for diagnostic and vaccine applications, it is imperative that efficient methods are developed to test the encoded proteins for immunological reactivity. In this study, we have used 220 synthetic peptides covering sequences of 12 open reading frames (ORFs) of RD1 and tested them as a single pool (RD1(pool)) with peripheral blood mononuclear cells obtained from pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects in Th1 cell assays that measure antigen-induced proliferation and IFN-gamma secretion. The results showed that RD1(pool) induced strong responses in both TB patients and BCG-vaccinated healthy subjects. The subsequent testing of peptide pools of individual ORFs revealed that all ORFs induced positive responses in a portion of donors, but PPE68, CFP10, and ESAT6 induced strong responses in TB patients and PPE68 induced strong responses in BCG-vaccinated healthy subjects. In addition, HLA-DR and -DQ typing of donors and HLA-DR binding prediction analysis of proteins suggested HLA-promiscuous presentation of PPE68, CFP10, and ESAT6. Further testing of individual peptides showed that a single peptide of PPE68 (121-VLTATNFFGINTIPIALTEMDYFIR-145) was immunodominant. The search for sequence homology revealed that a part of this peptide, 124-ATNFFGINTIPIAL-137, was present in several PPE family proteins of M. tuberculosis and M. bovis BCG vaccines. Further experiments limited the promiscuous and immunodominant epitope region to the 10-amino-acid cross-reactive sequence 127-FFGINTIPIA-136.     

IgA Response to ESAT‐6/CFP‐10 and Rv2031 Antigens Varies in Patients With Culture‐Confirmed Pulmonary Tuberculosis,Healthy Mycobacterium tuberculosis–Infected and Non‐Infected Individuals in a Tuberculosis Endemic Setting,Ethiopia

Little attention has been given to the role of antibodies against Mycobacterium tuberculosis (Mtb) infection. We have compared the levels of IgA and IgG against ESAT‐6/CFP‐10 and Rv2031c antigens in sera of patients with culture‐confirmed pulmonary tuberculosis (PTB), healthy Mtb‐infected and non‐infected individuals in endemic TB settings. Venous blood samples were collected from 166 study participants; sera were separated and assayed by an enzyme‐linked immunosorbent assay (ELISA). QuantiFERON‐TB Gold In‐Tube (QFTGIT) assay was used for the screening of latent TB infection. The mean optical density (OD) values of IgA against ESAT‐6/CFP‐10 and Rv2031 were significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.001). The mean OD values of IgG against ESAT‐6/CFP‐10 and Rv2031 were also significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.05). The mean OD values of IgA against both antigens were also higher in sera of healthy Mtb‐infected cases compared with non‐infected individuals. There were positive correlations (P < 0.05) between the level of IFN‐γ induced in QFTGIT assay and the OD values of serum IgA against both antigens in healthy Mtb‐infected subjects. This study shows the potential of IgA response against ESAT‐6/CFP‐10 and Rv2031 antigens in discriminating clinical TB from healthy Mtb‐infected and non‐infected cases. Nevertheless, further well‐designed cohort study is needed to fully realize the full potential of this diagnostic marker.     

Bacterial protein secretion is required for priming of CD8+ T cells specific for the Mycobacterium tuberculosis antigen CFP10

Mycobacterium tuberculosis infection elicits antigen-specific CD8(+) T cells that are required to control disease. It is unknown how the major histocompatibility complex class I (MHC-I) pathway samples mycobacterial antigens. CFP10 and ESAT6 are important virulence factors secreted by M. tuberculosis, and they are immunodominant targets of the human and murine T-cell response. Here, we test the hypothesis that CFP10 secretion by M. tuberculosis is required for the priming of CD8(+) T cells in vivo. Our results reveal an explicit dependence upon the bacterial secretion of the CFP10 antigen for the induction of antigen-specific CD8(+) T cells in vivo. By using well-defined M. tuberculosis mutants and carefully controlling for virulence, we show that ESX-1 function is required for the priming of CD8(+) T cells specific for CFP10. CD4(+) and CD8(+) T-cell responses to mycobacterial antigens secreted independently of ESX-1 were unaffected, suggesting that ESX-1-dependent phagosomal escape is not required for CD8(+) T-cell priming during infection. We propose that the overrepresentation of secreted proteins as dominant targets of the CD8(+) T-cell response during M. tuberculosis infection is a consequence of their preferential sampling by the MHC-I pathway. The implications of these findings should be considered in all models of antigen presentation during M. tuberculosis infection and in vaccine development.     

The CFP10/ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection

Tuberculosis is a human disease caused by infection with Mycobacterium tuberculosis. M. tuberculosis (Mtb) is a facultative intracellular pathogen. The alveolar macrophages provide a critical niche for the intracellular pathogen. It has been shown that virulent strains mycobacteria (Mtb-H37Rv, Mycobacterium bovis) induce less apoptosis in host macrophage than avirulent mycobacteria strains (Bacillus Calmette-Guérin, H37Ra). Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrary, it presents in all virulent strains of M. tuberculosis and M. bovis. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by RD1 genes Rv3874 and Rv3875, respectively. Recent studies indicated that the CFP10 and ESAT6 played an important role in M. tuberculosis virulence. It has been shown that incorporation of the RD1 region into BCG to restore the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence. Meanwhile, several studies showed that CFP10 and ESAT6 could inhibit and/or promote the production of tumor necrosis factor α (TNF-α) from macrophages. Furthermore, TNF-α can induce apoptosis and necrosis of infected macrophages in tuberculosis. Considering above results, we hypothesize that the CFP10 and ESAT6 may be involved in the virulence of Mycobacterium through modulating macrophage cell death.