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1.
11种牛分枝杆菌抗原在牛结核病诊断中的初步评价 总被引：1，自引：1，他引：0
为筛选及评价用于牛结核病诊断的抗原,本试验将CFP-10、ESAT-6、TB10.4、TB27.4、MPT51、MPT63、MPT64、MPB70、MPB83、Rv3872和Ag85B共11种牛分枝杆菌抗原分别作为包被抗原建立间接ELISA方法,比较其对牛结核病的检出率;同时利用豚鼠和牛的皮试试验评价重组蛋白作为皮试试验刺激原的潜力。此外,将重组蛋白分别刺激结核病阳性牛和阴性牛的抗凝血24h,检测血浆中的IFN-γ水平,评价各重组蛋白作为IFN-γ释放试验刺激原的潜力。结果显示,不同重组蛋白对结核病阳性血清的反应活性不一,MPB70总检出率最高,为59.7%;其次是Ag85B、ESAT-6和MPB83,检出率均在45%以上;MPT51的检出率最低,仅为2.2%。豚鼠和牛皮试试验均显示,单个重组蛋白作为刺激原难以产生令人满意的迟发型过敏反应(delayed type hypersensitivity,DTH),而TB10.4、TB27.4、MPT64、MPT63或Rv3872作为补充抗原,分别与CFP-10或ESAT-6混合,均可特异性地刺激结核病阳性牛产生较强的DTH反应,且与PPD-B无显著差异(P0.05)。重组蛋白CFP-10、ESAT-6、TB10.4和MPT51均能刺激结核病牛全血释放一定的IFN-γ,其中CFP-10、CFP-10-ESAT-6串联蛋白和MPT51刺激结核病阳性牛全血释放的IFN-γ显著高于阴性牛(P0.05)。因此,这11种牛分枝杆菌抗原并不适合单独用于牛结核病的血清学诊断、皮试试验或IFN-γ释放试验,但以CFP-10和ESAT-6为核心,TB10.4、TB27.4、MPT64、MPT63、Rv3872或MPT51作为其补充抗原,均能提高检测敏感性,有作为皮试试验和IFN-γ释放试验特异性刺激原用于牛结核病诊断的潜力。 相似文献
2.
低分子质量的蛋白抗原CFP-10是一种重要的牛分支杆菌早期分泌蛋白.为了检测该蛋白和其他几种抗原在牛结核病诊断中的临床应用,对CFP-10的基因进行克隆,鉴定,并在原核系统中表达.PCR法扩增cfp-10基因片段,连接到pET-22b( )原核表达载体中,再转入表达宿主E.coli BL21(DE3)PlysS菌株内,用IPTG诱导,进行蛋白表达、纯化.分别以CFP-10、ESAT-6、MPT83、MPT70、牛PPD、CFP-10与ESAT-6混合蛋白,MPT83与MPT70混合蛋白为抗原,用间接ELISA法诊断牛结核病.结果表明,以CFP-10与ESAT-6混合蛋白作为抗原检测牛结核病的特异性和敏感性分别达到了100%和63.6%,均超过了其他单抗原或抗原组合,为以筛选合适抗原为基础的血清学诊断技术提供了有力的支持并创造了良好的应用前景. 相似文献
3.
本研究通过融合表达Rv3872、CFP-10和ESAT-6蛋白,以改良牛结核病特异性鉴别诊断抗原CFP-10-ESAT-6,提高牛结核抗体鉴别诊断法的灵敏度。利用PCR方法,克隆牛分枝杆菌RD1区上述三个基因,通过酶切连接方法,将三个基因串联在pET-28a载体上,基因之间用Linker序列连接。融合基因在大肠杆菌内经IPTG0.4mmol/L诱导3h,目的蛋白经SDS-PAGE证实表达在上清中,Western-blot分析证实表达蛋白具有免疫原性。以所表达的融合蛋白作为包被抗原建立间接ELISA,检测了44份牛分枝杆菌感染背景清楚的临床奶牛血清。26份阳性血清中,Rv3872-CFP-10-ESAT-6融合蛋白ELISA检出阳性样本18份,检测灵敏度为69%,而CFP-10-ESAT-6融合蛋白ELISA只检出阳性样本4份。18份阴性血清中,两种ELISA均检出17份阴性血清,特异性都为94%（17/18）。因此,Rv3872-CFP-10-ESAT-6融合蛋白在对牛结核抗体检测特异性没有降低的情况下,敏感性获得了显著提高,这对牛结核病的早期鉴别诊断方法的建立具有重要意义。 相似文献
4.
为探索TB27.4蛋白在牛结核病鉴别诊断中的作用,本试验以牛分枝杆菌Vallee Ⅲ株基因组DNA为模板,PCR扩增tb27.4全长基因片段,将其定向克隆到原核表达载体pET-32a(+)中,构建重组质粒pET-TB27.4,优化原核表达条件,并用AKTA Purifier对蛋白的纯化条件进行优化。SDS-PAGE结果显示重组蛋白为可溶性表达,且大小与理论值相符,用牛分枝杆菌阳性血清进行Western blotting检测有特异性条带,且可特异性地刺激牛分枝杆菌感染牛外周血淋巴细胞释放大量IFN-γ。结果表明,重组蛋白TB27.4具有良好的B细胞活性和T细胞刺激活性,为进一步研究其在牛结核病诊断中的作用奠定了基础。 相似文献
5.
试验旨在评价细胞因子IL-6和IL-17 mRNA转录水平与牛分枝杆菌感染之间的关系,及其在牛结核病诊断中的应用潜力。通过皮内变态反应试验和IFN-γ释放试验临床筛选结核病阳性牛和结核病阴性牛,采集试验动物抗凝全血,分离、收集外周血淋巴细胞,分别用牛结核菌素（PPD-B）、禽结核菌素（PPD-A）、重组蛋白CFP-10-ESAT-6（CE）、pET-32a载体标签蛋白（PET）或PBS 37℃培养6 h,用实时荧光定量PCR检测细胞因子IL-6、IL-17和IFN-γ的mRNA相对转录水平。结果显示,PET和空白对照PBS类似,不能刺激细胞因子mRNA转录水平的提高,表明CE中包含的PET对试验的影响可忽略不计;牛外周血淋巴细胞经PPD-B、PPD-A或CE刺激后,结核病阳性牛样品中IL-17和IFN-γ的mRNA转录水平均显著高于结核病阴性牛（P<0.05）,其中PPD-B刺激效果强于CE和PPD-A,而CE刺激的特异性更好;选取CE作为最佳刺激源,结果显示,IL-17和IFN-γ的mRNA转录水平之间相关性良好（spearman r=0.79）,并初步建立了基于IL-17和IFN-γ转录水平的实时荧光定量PCR检测方法;以此方法对14头结核病阳性牛进行临床检验,IL-17实时荧光定量PCR法的阳性样本检出率为85.7%,高于IFN-γ（71.4%）。本研究结果初步表明,牛分枝杆菌特异性抗原（PPD-B、CE）诱导的IL-17 mRNA转录水平与牛结核病相关,以CE为刺激源建立的IL-17实时荧光定量PCR检测方法具有用于牛结核病诊断的潜力。 相似文献
6.
《中国兽医学报》2014,(9):1486-1490
CFP-10和ESAT-6是牛分枝杆菌的免疫优势抗原,可诱导机体产生IFN-γ,在牛结核病的免疫诊断中发挥重要作用。本试验分别克隆了牛分枝杆菌CFP-10和ESAT-6基因,并将这2个基因融合扩增,构建重组表达质粒pET28a/CFP-10-ESAT-6,重组质粒在大肠杆菌BL21中进行IPTG诱导表达。重组蛋白主要以分泌表达的形式存在于表达上清中,收集上清中的目的蛋白进行Ni亲和层析柱和分子筛两步纯化,蛋白纯度分别达到90%和95%。将纯化的重组蛋白以2mg/L的质量浓度包被酶标反应板,用间接ELISA方法检测不同来源牛血清中的结核病抗体,结果表明,表达的重组融合蛋白具有良好的抗原活性,可有效识别阴、阳性结核病血清。 相似文献
7.
为了研究牛结核病新型诊断抗原,试验根据GenBank中Mycobacterium bovis基因序列设计1对引物,将牛结核分枝杆菌MPB70基因构建到pET22b(+)原核表达载体上,将重组载体转到E.coliBL21(DE3)中,经IPTG诱导获得高效表达,并进行SDS-PAGE和Westen-blot分析。结果表明:MPB70蛋白以可溶形式在细胞周质中表达,有部分蛋白以包涵体形式在细胞质中表达,其分子质量约为30ku,蛋白表达量占菌体总蛋白的20%;重组MPB70蛋白可与牛分枝杆菌阳性血清发生特异性反应。说明重组MPB70蛋白能够作为诊断抗原。 相似文献
8.
IP-10作为牛结核病诊断标志物的初步探究 总被引：1，自引：0，他引：1
为评价细胞因子IL-12 p40、IP-10和TNF-α转录水平与牛分枝杆菌感染之间的关系,及其在牛结核病诊断中的应用潜力。采集田间筛选的结核病阳性牛、结核病阴性牛以及牛分枝杆菌68002人工感染牛的外周血淋巴细胞,经牛结核菌素(PPDB)、重组蛋白CFP-10-ESAT-6(CE)、MPT63、PET和PBS分别刺激6 h,提取细胞总RNA,用荧光定量PCR检测IL-12 p40、IP-10、IFN-γ和TNF-α的转录水平。结果显示结核病阳性牛的外周血淋巴细胞经PPDB和CE刺激后,其IP-10的m RNA转录水平显著高于结核病阴性牛,且与IFN-γ的m RNA转录水平具有良好的相关性;初步建立牛结核病IFN-γ和IP-10的Real-time PCR检测方法,其对临床阳性样本的检出率分别为71.45%和78.57%。因此,IP-10的m RNA转录水平与牛分枝杆菌的感染相关,有作为牛结核病诊断标志物的潜力。 相似文献
9.
牛结核杆菌MPB70-ESAT-6融合蛋白的原核表达及抗原反应性分析 总被引：1，自引：0，他引：1
为增强单个蛋白的抗原性,利用(Gly4Ser)3柔性连接肽将牛结核杆菌MPB70和ESAT-6融合。采用重叠延伸PCR技术将牛结核杆菌mpb70与esat-6基因连接,获得融合基因mpb70-esat-6,连接至T-Vector pMD19中,获得克隆质粒pMD-70-esat-6。经Bam HⅠ、EcoRⅠ酶切、纯化,并与p ET28a(+)载体连接,构建了pET-70-esat-6重组表达质粒。SDS-PAGE发现,在27.2 ku处表达了融合蛋白MPB70-ESAT-6,Western blotting证实,MPB70-ESAT-6与牛结核阳性血清反应性良好。MPB70-ESAT-6融合蛋白的研究为牛结核病诊断抗原及相关疫苗研究奠定了基础。 相似文献
10.
牛结核病抗体胶体金快速检测技术的建立和应用 总被引：10，自引：0，他引：10
为了建立一种快速检测牛分支杆菌抗体的新方法用于诊断牛结核病,利用胶体金免疫层析技术原理,用原核诱导表达的牛分支杆菌抗原蛋白MPB83和MPB70分别作为胶体金标记抗原和检测线上的捕获抗原,制备牛结核抗体检测试纸条.结果表明,粒径为40 nm的胶体金制备的试纸条敏感性最高,胶体金最佳标记pH为6.0,MPB83抗原最适标记量为每毫升胶体金6.5 μg,MPB70抗原的最适包被浓度为3.0 mg/mL,抗MPB83蛋白IgG的最佳包被浓度为2.5 mg/mL,交叉试验证明试纸条不与牛的其他非相关疾病的阳性血清反应,具有较高的特异性.比较试验证明其敏感性显著高于韩国进口试纸条.在上述试验条件下生产了一批胶体金试纸条进行临床样品检测,并与细菌分离培养、结核菌素皮内变态反应(TST)和韩国试纸条比较.本试纸条与牛分支杆菌分离培养的符合率为85%,与TST的符合率为79.73%,与韩国试纸条的符合率为98.75%.快速检测牛结核抗体的免疫层析试纸条具有敏感、特异、简便、快速的特点,适用于对牛结核病进行普查和检疫,也可作为TST的辅助诊断方法,在牛结核病根除计划中具有良好的应用前景. 相似文献
11.
Antigen specific immunological responses of badgers (Meles meles) experimentally infected with Mycobacterium bovis 总被引：2，自引：0，他引：2
Lesellier S Corner L Costello E Sleeman P Lyashchenko K Greenwald R Esfandiari J Singh M Hewinson RG Chambers M Gormley E 《Veterinary immunology and immunopathology》2008,122(1-2):35-45
European badgers (Meles meles) are considered to be an important reservoir of infection for Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. Accurate tests are required for tuberculosis surveillance in badger populations and to provide a basis for the development of strategies, including vaccination, to reduce the incidence of the infection. In this study, we have developed an endobronchial M. bovis infection model in badgers in which we measured cell-mediated immune and serological responses for up to 24 weeks post-infection. Groups of badgers were subjected to necropsy at 6-week intervals and the gross lesion severity status compared with immune responses measured in blood samples taken throughout the course of the study. The panel of antigens included bovine and avian tuberculins (PPD) as well as single antigens, ESAT-6, CFP-10, MPB70, Rv3019c, Rv3873, Rv3878 and Rv3879, all known to be recognised by the immune system in other animal models of tuberculosis infection. Our results demonstrated that M. bovis infected badgers responded to specific antigens as early as 6 weeks post-infection, consistent with the presence of visible lesions. The data also revealed unique patterns of antigen recognition with high levels of PBMC proliferation in the presence of CFP-10 but low proliferation levels with ESAT-6. Using a multi-antigen print immunoassay (MAPIA), we were able to confirm that MPB83 is the dominant antigen recognised by serum antibodies in infected badgers. 相似文献
12.
Schiller I Waters WR RayWaters W Vordermeier HM Jemmi T Welsh M Keck N Whelan A Gormley E Boschiroli ML Moyen JL Vela C Cagiola M Buddle BM Palmer M Thacker T Oesch B 《Veterinary microbiology》2011,151(1-2):153-159
Switzerland has been officially free of bovine tuberculosis (OTF) since 1960. Since 1980 the control of bovine tuberculosis (bTB) has been reduced to passive abattoir surveillance. Isolated cases of bTB, partly due to reactivation of human Mycobacterium bovis infections with subsequent transmission to cattle, have been noticed in the last years. In Europe, the overall prevalence of bTB is slightly increasing. Both OTF and non-OTF countries report increases in the proportion of bTB positive cattle herds. Current bTB eradication and control programs in Europe are facing a range of challenges. Whole herd depopulation is becoming a less attractive option for economic reasons and due to animal welfare concerns. Live animal trade is increasing both at national and international levels. Regarding these tendencies and taking into account the chronicity of bTB infection, pre-movement testing is becoming increasingly important as a central tool for eradication and for protection against re-introduction of bTB. Pre-movement testing, however specifically focuses on the infection status in individuals, requiring a high level of diagnostic accuracy to correctly diagnose infected animals. Current screening tests for bTB, however, have been designed to meet demands as herd tests. This illustrates that the modification of existing and/or the development of new diagnostics for bTB might be needed. The tuberculin skin test (TST), the primary screening test for bTB may in certain situations have low sensitivity. The interferon gamma (IFN-γ) assay is accepted to be more sensitive compared to TST. Reduced specificity, however, especially in areas of low bTB prevalence raises concerns. New antigen combinations including Rv3615c, OmpATb and others have been shown to complement ESAT-6 and CFP-10 in the whole blood IFN-γ assay and resulted in improved sensitivity (compared to ESAT-6 and CFP-10) and specificity (compared to tuberculins). Lesion detection after slaughter represents a cost-effective procedure for passive surveillance of bTB, especially in areas of low prevalence or in regions free of bTB; however, its sensitivity is very low. This illustrates that trade is linked with a certain risk to re-introduce bTB in OTF regions or countries and that there may be delays in detecting a re-introduction of bTB. In conclusion, regarding the fact that some parameters linked with bTB programs are changing, the development of improved diagnostic tests with a high reliability for use as individual animal tests will be important for future eradication of bTB, in line with international commitment to high standard animal health programs. 相似文献
13.
间接ELISA检测牛分枝杆菌抗体方法的建立及初步应用 总被引：6，自引：0，他引：6
针对现行牛结核病检疫方法结核菌素皮内变态反应(TST)的不足,本研究选用牛分枝杆菌特异性抗原MPB83、MPB70及CFP10与ESAT-6融合蛋白(CFP10-ESAT-6)分别建立了检测牛分枝杆菌特异抗体的间接酶联免疫吸附方法(ELISA)。上述3种抗原中一种以上抗原的特异性抗体为阳性时,即可判断为结核检测阳性。以TST为标准,判断ELISA方法的敏感性与特异性。结果证明,ELISA方法具有较好的敏感性(71.4%)。对ELISA检测为强阳性的奶牛进行细菌分离,并对5头结核菌分离阳性奶牛进行TST检测,结果有3头为TST阳性,2头可疑,显示出ELISA方法对严重感染牛的检测较TST具有更高的敏感性。由于TST与ELISA分别以细胞免疫与体液免疫为基础,两种检测方法结合应用可进一步提高牛结核病的检出率。 相似文献
14.
Use of mycobacterial peptides and recombinant proteins for the diagnosis of bovine tuberculosis in skin test-positive cattle 总被引：7，自引：0，他引：7
Buddle BM McCarthy AR Ryan TJ Pollock JM Vordermeier HM Hewinson RG Andersen P de Lisle GW 《The Veterinary record》2003,153(20):615-620
More accurate tests are required to test cattle which have reacted positively in the tuberculin skin test. For this purpose, a range of mycobacterial antigens, MPB59, MPB64, MPB70, MPB83, ESAT-6 and CFP10, were used either as recombinant proteins or as synthetic peptides in the whole blood interferon-gamma (IFN-gamma) test. Groups of uninfected cattle with typical 'non-specificity' problems were targeted, in particular animals with skin tuberculosis, animals vaccinated against Johne's disease and animals that were positive in the standard purified protein derivative (PPD)-based IFN-gamma test. The two study groups consisted of 74 Mycobacterium bovis-culture positive animals and 72 uninfected animals, all of which tested positive in the caudal fold tuberculin skin test eight to 28 days before the blood test. The use of combinations of ESAT-6 and CFP10 antigens, either as recombinant proteins or peptides, detected similar percentages of M bovis-infected animals as the PPD-based IFN-gamma test, but produced significantly fewer false positive reactions. The PPD-based IFN-gamma test was very effective in differentiating animals vaccinated against Johne's disease that were skin-test positive from those with bovine tuberculosis, and the use of PPD or specific mycobacterial antigens minimised the number of false positive reactions in animals with skin tuberculosis. 相似文献
15.
Assessment of defined antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle 总被引：17，自引：0，他引：17
Pollock JM Girvin RM Lightbody KA Clements RA Neill SD Buddle BM Andersen P 《The Veterinary record》2000,146(23):659-665
The continued use of purified protein derivative (PPD) tuberculin is considered to be the main factor which limits the specificity of diagnostic tests for bovine tuberculosis (TB). This study evaluated a whole blood interferon-gamma (IFN-gamma) assay and compared the diagnostic potential of PPD with two tuberculosis-specific antigens, ESAT-6 and MPB70. To provide estimates of sensitivity and specificity, responses were measured in 180 skin test-reacting cattle, of which 131 were confirmed as tuberculous, and in 128 cattle from TB-free herds. For the skin test reactors, there was a positive correlation between the IFN-gamma responses to PPD from Mycobacterium bovis (PPDB) and PPD from Mycobacterium avium (PPDA), indicating cross-reactivity between these complex antigens which are the basis of the skin test. In comparisons of the ESAT-6 IFN-gamma test with a PPD IFN-gamma test (using PPDB compared with PPDA), there was a decrease in sensitivity (76.3 per cent vs 89.3 per cent), but a clear increase in specificity (99.2 per cent vs 92.2 per cent). The provision of high specificity, even with lower sensitivity, offers major benefits for testing in areas with a low incidence of TB. 相似文献
16.
Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6 h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6 h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0+CD69+CD4+ memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium. 相似文献