莱姆病螺旋体鞭毛平截性蛋白的表达及其诊断潜力研究

目的原核表达伯氏疏螺旋体鞭毛蛋白Flagellin A基因特异性区段,获得重组鞭毛蛋白平截性蛋白作为诊断抗原,建立间接ELISA方法用于动物莱姆病的诊断。方法 PCR扩增获取伯氏疏螺旋体鞭毛蛋白基因的同源性较低的第394-798bp区段,构建重组质粒pGEX-4T-1/tFlaA,构建好的表达质粒转化到大肠杆菌BL21（DE3）中进行表达,并纯化重组蛋白,用纯化的表达蛋白作为莱姆病诊断的抗原,用于ELISA检测实验感染小鼠莱姆病。结果成功构建莱姆病螺旋体鞭毛平截性蛋白的表达载体,重组蛋白在宿主菌内高效、稳定表达,重组平截性蛋白显示了可作为ELISA诊断的抗原用于莱姆病的诊断价值。结论纯化的伯氏疏螺旋体鞭毛蛋白可作为莱姆病ELISA诊断抗原用于莱姆病的诊断,为莱姆病快速诊断试剂盒的开发打下基础。     

细粒棘球蚴特异性诊断抗原Eg-07279的制备及免疫原性研究

目的 筛选出包虫病特异性诊断抗原分子并验证其免疫原性。方法 对国家人类基因组南方研究中心公布的细粒棘球绦虫测序数据进行分析,利用生物信息学的方法筛选出六钩蚴中不表达且原头蚴中高表达的抗原分子Eg-07279,经重组克隆、表达后用亲和层析法纯化获得重组蛋白rEg-07279;重组蛋白免疫小鼠检测其特异性 IgG 水平并使用Western blot 验证其免疫原性。结果 筛选出的抗原分子Eg-07279经克隆、表达、纯化获得重组蛋白。利用该重组蛋白免疫小鼠,ELISA检测结果显示Eg-07279产生的特异性IgG水平(2.559 ± 0.125)明显高于空白组(0.319 0±0.01),差异有统计学意义(P<0.01);Western blot检测结果显示原头蚴继发感染组和重组蛋白免疫组血清均可识别该重组蛋白而空白对照组鼠血清不能识别。结论 获得细粒棘球蚴差异表达抗原Eg-07279并证实该重组蛋白具有较好的免疫原性,是较好的诊断抗原候选分子。     

双抗原夹心ELISA法检测登革病毒感染患者血清中的EDⅢ抗体

目的 建立一种检测登革病毒(dengue virus, DENV)特异性抗体的双抗原夹心ELISA方法?方法 利用毕赤酵母系统表达4个血清型登革病毒包膜蛋白Ⅲ区(envelope protein domain Ⅲ, EDⅢ),并采用改良过碘酸钠法对EDⅢ蛋白标记辣根过氧化物酶(Horseradish peroxidase, HRP),建立一种可同时检测4个血清型登革病毒EDⅢ蛋白特异性抗体的双抗原夹心ELISA法?并对2014年广州珠江医院门诊和住院确诊的登革热患者血清标本进行检测,并与澳洲Panbio MAC ELISA检测的敏感性进行比较?结果 本研究成功建立了一种检测4个血清型登革病毒EDⅢ蛋白特异性抗体的双抗原夹心ELISA法,该方法能同时检测到4个血清型登革病毒EDⅢ蛋白免疫的小鼠血清和Ⅰ型登革病毒EDⅢ蛋白免疫的兔血清,而与烟曲霉AF-MP蛋白免疫的小鼠血清和兔血清均无交叉反应?对184份健康人血清标本检测全为阴性,而对168份确诊为登革病毒感染患者血清标本检测结果表明,两种诊断方法检测结果差异无统计学意义,P<0.001(57.1% vs 57.7%),联合NS1抗原检测方法,其敏感性提高到97.6%?结论 双抗原夹心ELISA法检测登革病毒EDⅢ特异性抗体具有高度的特异性,但如果能同时联合抗原检测可明显提高登革热的早期诊断率?     

胶体金免疫层析法检测结核分枝杆菌特异性IgG/IgM抗体对结核病诊断的应用价值

目的 评价胶体金免疫层析法检测血清中结核分枝杆菌特异性IgG/IgM抗体在结核病诊断中的应用价值。方法 收集结核病患者和健康人的血清样本共332份及其背景资料,采用胶体金法检测血清中特异性结核抗体IgG/IgM,与临床诊断和细菌学检测结果比较,使用SPSS 22.0统计软件对结果进行比较分析,以P<0.05为差异具有统计学意义。结果 胶体金免疫层析法检测结核病患者中特异性结核抗体IgG/IgM的灵敏度为41.15%、特异性为91.67%,检测菌阳和菌阴结核病患者的敏感性分别为51.38%和33.77%。在全部结核病患者中,特异性结核抗体IgG/IgM检测检出率(41.15%)显著高于痰涂片法(18.84%)和痰菌培养法(36.15%)(P<0.05)。结核抗体检测、痰涂片法和痰培养法三种方法联合检测阳性率为61.54%,高于单种方法检测或其中两种方法联合检测。结论 胶体金免疫层析法检测血清中结核分枝杆菌抗体具有灵敏、特异、快速和简便等优点,可用于结核病的筛查,同时该方法也具有一定的局限性,敏感度和特异度有待进一步的提高,因此不可单独用于诊断结核病,可配合痰细菌学、影像学和临床表现等进行辅助诊断。     

布鲁氏菌鞭毛钩蛋白FlgE和融合蛋白BLS-FlgE的克隆、表达及免疫诊断研究

目的 分别构建含目的基因flgE和bls-flgE的原核表达载体,表达纯化重组蛋白,并对其免疫诊断价值进行初步评价。方法 从NCBI得到布鲁氏菌鞭毛钩蛋白基因flgE和2,4二氧四氢蝶啶合酶基因(bls)序列,设计引物,利用PCR技术扩增得到目的基因,与载体pET30a连接,转化E.coli BL21感受态细胞,IPTG诱导蛋白表达,SDS-PAGE及Western blot鉴定,使用带His标签的蛋白纯化试剂盒纯化蛋白。纯化后的蛋白建立ELISA法检测布病阳性血清和健康人血清,收集灵敏度和特异度数据。结果 成功克隆了FlgE鞭毛钩蛋白和BLS-FlgE融合蛋白,经过优化表达条件,目的蛋白获得较大量的表达,免疫印迹结果显示可被阳性病人血清识别。以FlgE蛋白构建ELISA法检测得到灵敏度和特异度分别为70.83%和41.67%,以BLS-FlgE蛋白构建ELISA法检测得到灵敏度和特异度分别为68.75%和52.08%。结论 成功表达布鲁氏菌鞭毛钩蛋白FlgE和融合蛋白BLS-FlgE,分别作为诊断抗原总体符合率低,免疫诊断价值较低。     

布鲁氏菌OMP25与OMP31蛋白的表达及其间接ELISA诊断试剂盒研制

目的 为建立布鲁氏杆菌间接ELISA检测方法。方法 本研究根据GenBank中已登录猪种布鲁氏菌S2株全基因组序列,分别针对omp25和omp31设计一对引物,分别扩增并回收573 bp与783 bp目的基因片段,将其分别克隆入pET-28a原核表达载体中,构建重组质粒pET-OMP25与pET-OMP31,进行酶切鉴定与基因序列测定后,转化BL21表达菌,经IPTG诱导表达,OMP25与OMP31蛋白主要以包涵体形式存在;将目的蛋白纯化,应用Western blot进行蛋白活性的检测。结果 纯化的目的蛋白具有良好的免疫学活性。以纯化的重组蛋白为包被抗原,在确定了OMP25与OMP31蛋白最佳配比为4:1、抗原最佳包被浓度为10 μg/mL、血清的最佳稀释倍数为1:50倍稀释、临界值为0.314的基础上,建立羊布鲁氏菌病抗体间接ELISA诊断方法并组装成诊断试剂盒,并将试剂盒进行了特异性、敏感性、重复性、符合率试验、血清交叉反应性等检测。结论 研制的羊布鲁氏菌病抗体ELISA诊断试剂盒具有良好的特异性、敏感性,可重复性,可应用于临床样本检测。     

结核分枝杆菌Rv3425-Rv1168c蛋白融合表达与血清学评价

目的 原核表达结核分枝杆菌Rv3425-Rv1168c融合蛋白并进行纯化,通过酶联免疫吸附试验(ELISA)方法评价重组蛋白在结核病血清学诊断中的价值。方法 以结核分枝杆菌H37Rv基因组为模板,通过重叠PCR扩增得到Rv3425-Rv1168c全核酸序列克隆至表达载体pET-24b中,转入大肠杆菌BL21(DE3)进行诱导、表达和纯化,通过ELISA检测100份确诊结核病人、20例非结核呼吸疾病患者、100份健康人血清,评价融合蛋白进行临床血清学诊断的可行性。结果 Rv3425-Rv1168c融合蛋白在原核系统内获得高表达,纯化的融合蛋白经ELISA方法测定,在血清学实验中敏感性为54%,特异性为95%。结论 Rv3425-Rv1168c融合蛋白具有较高的抗原特异性和免疫原性,在结核病血清学诊断方面具有很大的应用价值。     

内蒙古大兴安岭林区牙克石段的蜱种及蜱携病原体感染调查

目的 调查内蒙古东部大兴安岭林区牙克石段的蜱种分布及病原体感染状况。方法 2016-2019年,在东部林区蜱活跃高峰期(5-6月)采用布旗法采集游离蜱标本,使用体视显微镜对蜱初步分类;提取蜱全基因组DNA,采用斑点热立克次体、无形体属、埃立克体属、莱姆病螺旋体和新型回归热螺旋体病原体的特异性基因进行PCR检测。结果 本次共采集蜱虫2 786只,通过体视显微镜和基因检测分析,隶属于1科3属4种,分别为全沟硬蜱(Ixodes persulcatus)、嗜群血蜱(Haemaphysalinae concinna)、日本血蜱(Haemaphysalinae japonica)、森林革蜱(Dermacentor silvarum)。其中,全沟硬蜱(78.6%,2 191/2 786)、嗜群血蜱(15.9%,442/2 786)为本地区的优势蜱种。在大兴安岭林区牙克石段的7个采样地区的蜱中,乌奴耳镇斑点热立克次体检出率最高,为74.2%(222/299);博克图镇无形体属检出率较高,为18.9%(39/206);库都尔镇埃立克体属、莱姆病螺旋体、新型回归热螺旋体的检出率均较高,分别为26.1%(12/46)、76.1%(35/46)、13.0%(6/46)。除新型回归热螺旋体外,其他4种病原体的检出率雄蜱均高于雌蜱(P<0.05);成蜱的无形体、埃立克体、莱姆病螺旋体检出率均高于幼蜱(P<0.05)。大兴安岭林区牙克石段蜱的复合感染率为19.5%(544/2 786)。其中,免渡河镇蜱携2种病原体复合感染率为40.6%(186/458),乌奴耳镇蜱携3种及以上病原体复合感染率分别是47.4%(36/76)、90%(9/10)。结论 大兴安岭林区牙克石段共存在4种蜱,蜱携莱姆病螺旋体最普遍,其中免渡河镇和乌奴耳镇的蜱普遍携带多种病原体,需重点加强该地区蜱媒传染病的监测和预防。     

重组结核分支杆菌38kD和16kD蛋白用于结核病血清学诊断的价值

目的　探讨国产重组结核分支杆菌蛋白38kD(rTPA38)和16kD(rTPA16)用于结核病的诊断价值。方法 以rTPA38和rTPA16为抗原,PPD为对照,用ELISA法检测血清中特异性抗结核抗体。结果 246例肺结核病组,rTPA38、rTPA16和PPD检测的灵敏度分别为66.3%、63.0%和72.3%。健康献血组、非结核呼吸疾病组和卡介苗接种阳转组血清同时用rTPA38、rTPA16和PPD检测,rTPA38特异性分别为97.6%、96.8%、86.0%。rTPA16特异性分别为94.7%、93.1%、75.0%。PPD特异性为93.4%、85.7%、67.9%。统计分析显示,rTPA38蛋白和PPD检测非结核呼吸疾病组,其阳性率有显著性差异 (P＜0.05)。两者检测卡介苗接种阳转组阳性率和血清抗体滴度有显著性差异 (P＜0.05)。rTPA38和rTPA16同时检测108例肺结核病组血清可提高11.1%的阳性率。结论 rTPA38蛋白抗原有较好的灵敏度和较高特异性,是ELISA的可靠抗原,与rTPA16联用可提高灵敏度,对结核病血清学诊断有较高参考价值。     

布鲁氏菌Omp2b优势T-B联合抗原表位的免疫应答特点研究

目的 通过生物信息技术预测分析Omp2b蛋白的优势B细胞表位和T细胞表位,筛选T-B联合抗原表位,探讨其免疫原性和激发免疫应答的特点,为开发有效的布鲁氏菌疫苗奠定基础。方法 1)利用生物信息学软件ProtParam,SOMPA,SWISS-MODEL,Rasmol,DNAStar,SYFPEITHI和IEDB来分析Omp2b蛋白的结构,预测T细胞和B细胞的优势表位以及T-B联合表位;2)ELISPOT法检测细胞中IFN-γ阳性细胞数;3)ELISA检测布鲁氏菌病患者血清中针对Omp2b蛋白的T-B联合肽的特异性IgG抗体水平。体外培养免疫细胞,检测上清液中的穿孔素和颗粒酶B。结果 生物信息软件综合分析Omp2b蛋白并筛选出了1个T-B细胞联合抗原表位的潜在区段196-216;患者组中分泌IFN-γ细胞数(SFU)较健康对照组增高(F=25.413,P<0.01),布鲁氏菌病患者血清中的IgG抗体、穿孔素和颗粒酶B的浓度也增加(F=13.653,P<0.01)。结论 Omp2b蛋白的T-B联合抗原表位可以产生特异性的体液免疫应答和细胞免疫应答,为布鲁氏菌表位疫苗的筛选与构建提供参考。     

Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates

In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent.     

Antigenic conservation of an immunodominant invariable region of the VlsE lipoprotein among European pathogenic genospecies of Borrelia burgdorferi SL

Lyme disease is caused by genetically divergent spirochetes, including 3 pathogenic genospecies: Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Serodiagnosis is complicated by this genetic diversity. A synthetic peptide (C(6)), based on the 26-mer invariable region (IR(6)) of the variable surface antigen of B. burgdorferi (VlsE), was used as ELISA antigen, to test serum samples collected from mice experimentally infected with the 3 genospecies and from European patients with Lyme disease. Regardless of the infecting strains, mice produced a strong antibody response to C(6), which indicates that IR(6) is antigenically conserved among the pathogenic genospecies. Twenty of 23 patients with culture-confirmed erythema migrans had a detectable antibody response to C(6). A sensitivity of 95.2% was achieved, with serum samples collected from patients with well-defined acrodermatitis chronica atrophicans. Fourteen of 20 patients with symptoms of late Lyme disease also had a positive anti-IR(6) ELISA. Thus, it is possible that C(6) may be used to serodiagnose Lyme disease universally.     

Diagnosis of lyme borreliosis in europe

In Europe, Lyme borreliosis is caused by at least three species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. Thus microbiological diagnosis in European patients must consider the heterogeneity of Lyme disease borreliae for development of diagnostic tools such as PCR primers and diagnostic antigens. According to guidelines of the German Society of Hygiene and Microbiology, the serological diagnosis should follow the principle of a two-step procedure. A sensitive ELISA differentiating IgM and IgG is recommended as the first step. In case the ELISA is reactive, it is followed by immunoblots (IgM and IgG) as the second step. The reactive diagnostic bands should be clearly identified, which is easy if recombinant antigens are used. The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens instead of whole cell lysates. Improved sensitivity resulted from use of recombinant proteins that are expressed primarily in vivo (e.g., VlsE) and combination of homologous proteins from different strains of borrelia (e.g., DbpA). It also appears promising to use recombinant proteins (DbpA, VlsE, others) or synthetic peptides (the conserved C6 peptide derived from VlsE) as ELISA antigens. At present, detection rates for serum antibodies are 20-50% in stage I, 70-90% in stage II, and nearly 100% in stage III Lyme disease. The main goals for the future are to improve specificity in general and sensitivity for diagnosis of early manifestations (stage I and II). Detection of the etiological agent by culture or PCR should be confined to specific indications and specialised laboratories. Recommended specimens are skin biopsy specimens, CSF and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50-70%) and synovial tissue or fluid (50-70% with PCR). CSF yields positive results in only 10-30% of patients. Methods that are not recommended for diagnostic purposes are antigen tests in body fluids, PCR of urine, and lymphocyte transformation tests.     

Evaluation of recombinant line immunoblot for detection of Lyme disease in Slovakia: comparison with two other immunoassays

In the present study the sensitivity and the specificity of three serological tests (enzyme-linked immunosorbent assay [ELISA], indirect fluorescent antibody test [IFA], and recombinant line immunoblot) were compared by examining 74 sera from patients diagnosed with Lyme disease in Eastern Slovakia. In addition, the reactivity to each of the recombinant proteins in the immunoblot was examined in order to evaluate their diagnostic value. Generally, the immunoblot (93.2%) and the ELISA (90.5%) were significantly more sensitive than the IFA (64.9%; df = 1; p < or = 0.001). Correlation between results of the ELISA, IFA, and immunoblot for IgM or IgG, when two tests were always compared, one to the other, ranged from r(s) = 0.673 to r(s) = 0.905. In the immunoblot, the highest sensitivity was observed in DbpA and VlsE proteins (76.9% and 84.6%, respectively) in IgG testing of the sera from the patient group of Lyme arthritis. VlsE proteins, together with OspC proteins, were also shown to be useful for IgM antibody detection in erythema migrans patients (up to 44.4% and 53.7% sensitivity, respectively). Our results indicate that both the ELISA and the recombinant immunoblot test were more satisfactory for seroconfirmation of Lyme disease than IFA. Moreover, the reseach confirmed diagnostic value of the in-vivo expressed proteins (VlsE and DbpA), which might have the potential to play an important role in improving whole-cell antigen-based testing.     

Lyme borreliosis

Lyme borreliosis is a multi-organ infection caused by spirochetes of the Borrelia burgdorferi sensu lato group with its species B burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, which are transmitted by ticks of the species Ixodes. Laboratory testing of Lyme borreliosis includes culture, antibody detection using ELISA with whole extracts or recombinant chimeric borrelia proteins, immunoblot, and PCR with different levels of sensitivity and specificity for each test. Common skin manifestations of Lyme borreliosis include erythema migrans, lymphocytoma, and acrodermatitis chronica atrophicans. The last two conditions are usually caused by B garinii and B afzelii, respectively, which are seen more frequently in Europe than in America. Late extracutaneous manifestations of Lyme borreliosis are characterised by carditis, neuroborreliosis, and arthritis. We present evidence-based treatment recommendations for Lyme borreliosis and review the prevention of Lyme borreliosis, including the Lyme vaccines.     

两种梅毒血清学检测方法的联合运用分析

目的进一步提高酶联免疫吸附试验(ELISA)对梅毒的筛查率,确保梅毒血清学的检测效果。方法采用ELISA联合甲苯胺红不加热血清试验(TRUST),对2011年上海浦东新区2 686例监测人群的血清样本进行梅毒血清学筛查,联合检测的血清学阳性样本用梅毒螺旋体明胶凝集试验(TPPA)甄别其生物学假阳性;再从2 397份两种方法均为阴性的样本中,随机抽取1 000份进行TPPA检测,甄别其生物学假阴性。结果 ELISA法相对于TPPA法的敏感性为98.9%,特异性为98.4%,两法间差异无统计学意义(P>0.05);ELISA与TRUST法联合应用后的敏感性接近100%,特异性为98.3%。结论 ELISA与TRUST方法的联合应用,其敏感性接近100%,表明其可用于梅毒筛查;另一方面,两者联合运用的特异性为98.3%,具有一定的假阳性率,表明其阳性样本(两法均为阳性的样本除外)还需应用TPPA方法进行最终确认。     

Enzyme-linked immunosorbent assay and indirect immunofluorescence assay for Lyme disease

The sensitivity and specificity of an indirect immunofluorescence assay (IFA) and ELISA for Lyme disease were estimated. Sera from patients with Lyme disease, patients with other infections, and healthy individuals were examined. Significant cross-reactivity occurred only with sera from patients with syphilis, yaws, and pinta . All tested sera from patients with Lyme disease, however, gave negative results in the rapid reagin screening test and the microhemagglutination assay for antibodies to Treponema pallidum confirmatory for syphilis. When sera from patients with treponemal diseases were excluded from the analysis, the IFA and ELISA were highly specific, having 97% and 100% reliability, respectively. Sensitivity of both tests varied with the stage of disease but was 100% for both tests during complicated Lyme disease. The results indicate that both tests are highly specific and sensitive for complicated Lyme disease but relatively insensitive for patients with erythema chronicum migrans alone.     

Growth-inhibiting antibody responses of humans vaccinated with recombinant outer surface protein A or infected with Borrelia burgdorferi or both

Serial serum samples from a 2-year human trial of outer surface protein (Osp) A vaccine were analyzed by Borrelia burgdorferi growth-inhibition assay (GIA) and anti-OspA ELISA to assess the antibody responses of vaccine recipients and subjects with Lyme disease. Although 74% of OspA recipients had a reciprocal GIA titer >/=64 after 3 vaccinations, none of the placebo recipients, even those with Lyme disease, had a GIA titer this high. The correlation between GIA and ELISA titers after 3 doses of vaccine was.84; however, more vaccine recipients had an elevated ELISA titer paired with low GIA titer than had a low ELISA titer with a high GIA titer. OspA-vaccine recipients who acquired Lyme disease had significantly lower serum GIA and ELISA titers after 3 immunizations than did age- and sex-matched OspA recipients without Lyme disease. Thus, vaccinated subjects had antibodies to native antigen on viable cells, and antibody assays with this specificity may predict protection of vaccinees against infection.