侵袭性烟曲霉感染特异性甘露聚糖捕获ELISA和rDNA ITS区DNA检测方法的建立及评价
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摘要
侵袭性曲霉病(invasive aspergillosis, IA)是一种主要由肺部感染播散至全身多个脏器的系统性感染疾病,是免疫受损人群的主要致死性并发症之一。腐生性丝状真菌烟曲霉(Aspergillus Fumigatus)是引起IA的最主要曲霉物种,这主要是由烟曲霉的孢子性质决定的,烟曲霉的孢子产量非常大,在环境中无处不在,平均每人每天可吸入上百个烟曲霉孢子,同时,其孢子体积小,易发生侵入性感染,且吸附能力强,生长速度快,对不良环境的抵抗力强。对于健康个体,吸入的孢子可及时被清除,但是在某些免疫受损患者群中,吸入的烟曲霉孢子可在肺部萌发、繁殖,引发肺曲霉病。孢子一旦进入血液循环,则可在其它组织器官定植,造成系统性全身感染,即IA。随着免疫抑制剂的使用,器官移植、造血干细胞移植、肿瘤化疗等治疗手段的出现,以及继发件免疫缺陷疾病如AIDS等的出现,导致大量易受烟曲霉感染的免疫缺陷人类宿主出现。对这些免疫缺陷个体,IA的感染率可高达50%。即使采用抗真菌治疗,死亡率也常常有50%之多。早期快速诊断有助于及时实施抗真菌治疗,控制病情,改善预后。然而,在大多数情况下,诊断问题尚不能解决,延误了有效的医学治疗。
现阶段,烟曲霉感染的主要诊断方法包括传统诊断、基因检测、特异性抗原抗体检测等。传统的诊断方法主要有影像学、无菌腔液标本培养和组织病理学鉴定等。影像学上的新月征(air crescent)和晕轮征(halo sign),由于类似特征在念珠菌病、军团菌病、巨细胞病毒感染、Kaposi肉瘤等患者体内也可观察到,并不能作为IA确诊的依据。呼吸道分泌物培养诊断敏感性低,而血培养即使在传播性感染中,也多表现为阴性。此外,标本培养还存在培养周期长等缺点。而组织病理学检查具有创伤性,并不适用于所有的患者。因此,传统的诊断方法已难以满足IA诊断的临床需要。
IA病情发展迅速,机体并无足够产生抗体的时间,以及免疫受损患者特异性免疫应答水平低的特点,使得抗体检测易出现假阴性结果。同时,每天吸入空气中大量烟曲霉孢子,正常个体机体内所存在的烟曲霉特异抗体,又可导致假阳性结果的出现。因此,单一的特异性抗体检测在IA诊断中的应用价值一直存在争议。近年来,IA的诊断学研究热点主要集中在抗原捕获和基因检测方面。
目前被国际认可的具有诊断价值的真菌抗原主要有两种：半乳甘露聚糖抗原(Galactomannan, GM)和(1,3)-p-D葡聚糖抗原(p-D glucan, BG),这两种检测方法已被美国和欧洲许多国家批准用于侵袭性真菌感染诊断中。然而,这两种方法具有敏感性和特异性均不稳定的应有局限性。如GM检验不能区分曲霉菌与马尔尼菲青霉菌的感染,且在病情发展中很快被清除掉,导致出现假阴性结果。BG检测(G试验)没有种属特异性,不能区分曲霉与酵母菌感染,对静脉使用白蛋白或γ球蛋白患者,抗肿瘤药物如香菇菌多糖使用患者,以及粘菌素E、头孢噻肟等抗菌药物使用患者的检测,均存在出现假阳性结果的可能。寻找新的特异性抗原靶标,建立灵敏度和特异性俱佳的抗原捕获检测方法在目前仍显得尤为重要。
随着分子生物学技术的广泛应用,烟曲霉临床分离菌株Af293的全基因组测序完成,很多研究工作者把目标转向根据基因组信息设计具有种、属特异性的引物,发展简便、快速的IA诊断方法。尽管基因检测方法要成为诊断IA的常规检测方法还存在很多问题,包括方法学标准化困难,对实验场地的配置、实验操作规范性、对人员的要求高等。基因检测仍具有其它诊断方法不可比拟的优点,包括可准确分类到菌株类型,获得感染物种耐药性等相关信息。因此,建立IA患者烟曲霉特异基因检测标准化方法,有助于实现IA的早期快速精确诊断。
由此,本研究的目的主要在于,建立烟曲霉感染特异性抗原捕获ELISA检测方法,同时建立烟曲霉感染基因检测标准化诊断方法,以求最终实现烟曲霉感染的早期诊断。研究内容主要有：
1.重组Afmp1p蛋白的毕赤酵母表达,抗rAfmp1p单克隆抗体的制备以及烟曲霉感染特异性Afmplp抗原捕获ELISA方法的建立与评价
Afmplp由AFMP1基因所编码,为烟曲霉半乳甘露聚糖蛋白,由本实验室与香港大学袁国勇教授等人合作,于2001年通过生物信息学分析发现。在发现该靶标蛋白后,使用大肠杆菌表达系统表达重组GST-Afmplp融合蛋白,制备抗GST-Afmplp豚鼠血清,使用多克隆抗体(polyclonal antibody, PAb)夹心法,建立了烟曲霉感染抗原捕获ELISA检测方法。然而,该方法对烟曲霉感染IA患者检测的灵敏度仅为53%,并不能达到临床样本检测的要求。这可能是由于多克隆抗体小身所具有的缺陷造成的,包括不同批次不同动物间不可避免的血清差异。为了弥补多克隆抗体所存在的缺陷,本实验室在之前的研究中,使用GST-Afmp1p免疫小鼠,制备抗GST-Afmplp单克隆抗体(monoclonal antibody, MAb),获得了较为理想的抗原夹心捕获单克隆抗体对,可检测至0.1ng/ml的重组蛋白。遗憾的是,该方法并不能捕获天然存在的Afmp1p抗原。这可能缘于大肠杆菌表达重组GST-Afmplp蛋白由于缺乏后期修饰,与天然Afmp1p蛋白间存在构象差异,从而造成的抗原表位改变。
在本研究中,为了弥补原核表达系统所存在的无后期加工等缺陷,选择pPICZ α-B表达载体,使用野生型毕赤酵母X33菌株,表达AFMP1基因,获得了与天然蛋白更为接近的重组Afmp1p蛋白(rAfmp1p).使用rAfmp1p免疫小鼠,制备抗rAfmp1p单克隆抗体,对其进行HRP标记,鉴定亚型,并通过竞争抑制ELISA分析其识别的表位类型。在这些研究的基础上,将单克隆抗体两两配对,进行重组蛋白(10ng/μ1)夹心捕获以及感染动物血清10倍稀释液夹心捕获筛选,获得最为理想的MAb抗体对。使用所获得的最佳抗体对,对重组蛋白倍比稀释液、多种真菌培养上清稀释液以及真菌感染兔血清稀释液进行夹心检测,从而对Afmp1p抗原捕获ELISA检测方法的灵敏度与特异度进行评价。
2.重组Afmp4p蛋白的毕赤酵母表达,抗rAfmp4p MAb的制备以及烟曲霉感染特异Afmp4p抗原捕获ELISA方法的建立与评价
AFMP4基因所编码Afmp4p蛋白为Afmp1p同源蛋白,由香港大学袁国勇教授等人通过生物信息学分析发现。在Afmp1p做为烟曲霉感染临床诊断标志物的意义尚未确定之时,对Afmp4p可能的诊断意义进行分析,可增加成功建立烟曲霉感染抗原捕获ELISA检测方法的几率。因此本研究在建立Afmp1p抗原捕获ELISA检测方法的同时,同步构建pPIC9K-AFMP4重组表达载体,使用毕赤酵母表达系统表达AFMP4基因,获得重组Afmp4p蛋白(rAfmp4p),并进一步制备鼠源性抗rAfmp4p单克隆抗体,进行表位分析与亚型鉴定后,将单克隆抗体两两配对,进行重组蛋白(10ng/μ1)夹心捕获以及感染动物血清10倍稀释液夹心捕获筛选,获得最为理想的单克隆抗体对。对所获得的抗体对,使用重组蛋白倍比稀释液、多种真菌培养上清稀释液以及真菌感染兔血清稀释液夹心检测,评价方法灵敏度与特异度。
3.烟曲霉感染rDNA ITS区巢式real-time PCR检测方法的建立
核糖体DNA (ribosomal DNA, rDNA)既有保守区又有可变区,在进化速率上较为保守,作为种级以上阶元的良好标记,广泛应用于物种系统学研究中。rDNA基因簇从5’到3’端依次为18S rDNA,内转录间隔区1(internal transcribed space, ITS1),5.8S rDNA, ITS2,28S rDNA,其中,ITS Ⅰ和ITS Ⅱ常被合称为ITS区。18S rDNA序列中既有保守区又有可变区,在进化速率上较为保守,是系统发育中种级以上阶元的良好标记。ITS区由于不加入成熟核糖体,受到的选择压力较小,进化速率较快,在绝大多数的真核生物中展现出极为广泛的序列多态性,表现为种内相对一致,种间差异较明显,能真实反映属间、种间以及菌株间的碱基对差异。因此,不同物种间18S rDNA和ITS区域的序列差异常用于物种鉴别研究中。
在本研究中,选取针对真菌rDNA ITS区通用引物1对(ITSS:5'-GTGAATCAT CGAATCTTTGAAC-3', ITSR:5'-TCCTCCGCTTATTGATATGC-3'),针对真菌18S rDNA区域通用引物1对(18SS:5'-ATTGGAGGGCAAGTCTGGTG-3',18SR:5'-CCGATCCCTAGTCGGCATAG-3'),对真菌基因组DNA、烟曲霉感染兔组织(肝、肾、肺)DNA、黄曲霉感染兔组织(肝、肾、肺)DNA以及模拟真菌败血症血液标本DNA扩增,判断两种方法的检测范围。同时对曲霉基因组DNA扩增产物直接测序,经序列比较分析,选择出具有物种特异性的检测靶标序列。为了弥补通用引物扩增后,产物需进一步测序才能鉴别到物种的缺点,对不同真菌的该段序列进行比较分析,设计曲霉特异real-time PCR引物与探针,尝试建立曲霉real-time PCR检测方法。为了弥补曲霉real-time PCR检测方法灵敏度低的缺陷,进一步建立巢式real-time PCR检测。
小结
通过以上三部分的工作,本研究所获得结果主要有：
1、在毕赤酵母中成功表达并纯化rAfmp1p,获得的rAfmp1p约为40kDa,略大于预期的31.4kDa,这可能是由于蛋白糖基化造成的。使用rAfmp1p免疫小鼠,并制备属源性单克隆抗体,共获得20株抗rAfmp1p单克隆抗体,分别以Afmp1-M1至Afmp1-M20命名。亚型分析发现,除Afmp1-M1外,所有的单克隆抗体均为IgG1.表位分析发现,这20株单克隆抗体可识别6种不同的抗原表位。将这些单克隆抗体两两配对,对rAfmp1p (10ng/μ1)以及烟曲霉感染兔血清10倍稀释液双抗体夹心检测,筛选获得了特异性高,灵敏度好的单克隆抗体对,建立了烟曲霉感染Afmp1p抗原捕获ELISA检测方法。其中,包被抗体为Afmp1-M3(包被浓度为10μg/ml),捕获抗体为Afmp1-M6-HRP.该Afmplp抗原捕获ELISA检测方法对rAfmp1p检测的灵敏度为400pg/ml,可捕获到烟曲霉1640培养上清16倍稀释液中的Afmp1p抗原,可检测到烟曲霉感染24h后兔血清16倍稀释液中的Afmp1p抗原。对其它曲霉感染兔血清以及其它病原真菌1640培养上清的检测,均表现为阴性。
2、在毕赤酵母中成功表达并纯化rAfmp4p,该蛋白约为20kDa,与预期大小吻合。使用rAfmp4p免疫小鼠并制备属源性单克隆抗体,共获得13株抗rAfmp4p单克隆抗体,以Afmp4-M1至Afmp4-M13命名。亚型分析发现,所有的单克隆抗体均为IgGl。表位分析发现,这13株单克隆抗体可识别5种不同的抗原表位。将这些单克隆抗体两两配对,对rAfmp4p (10ng/μ1)以及烟曲霉感染兔血清10倍稀释液双抗体夹心检测,筛选获得了特异性高,灵敏度好的单克隆抗体对,建立了烟曲霉感染Afmp4p抗原捕获ELISA检测方法。其中,包被抗体为Afmp4-M9(包被浓度为10μg/ml),捕获抗体为Afmp4-M2-HRP。该Afmp1p抗原捕获ELISA检测法对rAfmp4p检测的灵敏度为800pg/ml,在烟曲霉1640培养上清512倍稀后,仍可捕获到释放到培养上清中的Afmp4p抗原,可检测出烟曲霉感染24h后兔血清8倍稀释液中的抗原。对其它曲霉感染兔血清以及其它病原真菌1640培养上清的检测,均表现为阴性。
3、使用真菌18S rDNA通用引物(18SS、8SR),和真菌rDNA ITS区通用引物(ITSS、ITSR),对真菌基因组DNA、烟曲霉感染兔组织(肝、肾、肺)DNA、黄曲霉感染兔组织(肝、’肾、肺)DNA以及模拟真菌败血症血液标本DNA的扩增,均表现为阳性。对扩增产物的直接测序以及Blast分析发现,扩增出的18S rDNA片段序列(504bp)为真菌共有片段,并无物种特异性,扩增出的rDNA ITS序列(299bp)具有物种特异性,不同真菌之间,以及曲霉属内不同曲霉之间均存在差异,属内物种序列相似性小于92%。使用rDNA ITS区通用引物PCR扩增与直接测序相结合,可成功对不同的真菌物种进行区分鉴别。通过不同真菌rDNA ITS区的比较分析,设计real-time PCR引物,其中上游引物为5'-TATGGGGCTTTGTCACCTC-3',下游引物为5'-TCCTCCGCTTA TTGATATG-3', Taqman特异探针为5'-CCGGCGCCAGCCGACACCCAACTTTA-3’。该方法对烟曲霉、黄曲霉以及土曲霉DNA检测表现为阳性,然而ct值高,灵敏度低。由于real-time PCR引物扩增片段区域位于ITS通用引物扩增区域内部,因此建立使用ITS通用引物第一轮扩增,real-time PCR引物第二轮扩增并荧光检测的巢式real-time PCR检测方法。该方法最适第一轮扩增循环次数为15,该方法可成功检测烟曲霉、黄曲霉以及土曲霉DNA,烟曲霉、黄曲霉感染兔组织(肝、肾、肺)DNA,以及模拟真菌败血症血液标本DNA。而对其它真菌,包括构巢曲霉、黑曲霉、马尔尼菲青霉、念珠菌、以及新生隐球菌DNA的检测,均表现为阴性。
总之,本研究成功建立了3种烟曲霉感染检测方法,其中2种为烟曲霉特异性甘露聚糖抗原捕获检测方法,1种为曲霉特异性巢式real-time PCR检测方法。遗憾的是,由于临床样本的缺乏,这些方法在临床上的应用价值,尚需进一步实验确认。
Aspergillus fumigatus is a ubiquitous filamentous fungus in the environment. On average, humans inhale hundreds of Aspergillus conidia daily. These conidia are eliminated efficiently by innate immune mechanisms in immunocompetent hosts, but could germinate and colonize in immunocompromised host. As a result of the increasing use of transplantation for end-organ disease, the use of immunosuppressive and myeloablative therapies for autoimmune and neoplastic diseases, and the human immunodeficiency virus (AIDS) pandemic, the number of immunocompromised patients is steadily expanding. In these patients, the conidia of A. fumigatus, with diameters (3to5μm) that allow them to traverse the terminal respiratory airways and reach the pulmonary alveoli, could escape the defense line and germinate in the lung, causing pulmonary aspergillosis. In most cases, the fungal pathogen will also migrate to other organs, such as myocardium, kidney, liver, spleen, soft tissue, and bone, causing invasive aspergillosis (IA)--a severe and usually fatal systematic infection. The incidence of IA can reach as high as50%in acute leukemia if the patients have a risk of Aspergillus propagation and dissemination, such as during building/ construction work. The mortality rate is often around50%. The eary diagnosis is very impotant for IA patients。
Since the signs and symptoms of IA are nonspecific, early clinical diagnosis of the infection is often difficult. The "gold standard" for diagnosis is to obtain a positive culture of A. fumigatus and to obtain histological evidence of mycelial growth in biopsy specimens. However, owing to its invasiveness, biopsy is often precluded from patients in critical conditions. A. fumigatus cultures from blood or respiratory specimens are often false-negative, especially during early stages of the disease. As an adjunctive measure to microbiological methods, antibody detection is usually performed for the diagnosis of IA, but these assays are usually insufficient owing to limits in their sensitivity and specificity. False-negative results are often come out because of the fulminant nature of the disease and the poor immunological status of the host, and false-positive results also exist in healthy individuals because of the prolong interaction with conidia. At present, most of the studies focused on the identification of antigenic markers and the DNA targets of IA.
At present, the diagnostic value of galactomannan (GM), a major component of the Aspergillus cell wall, has been identified. Platelia Aspergillus (Bio-Rad, Marnes-La-Coquette, France) assay, an immunoassays that employ a rat immunoglobulin M (IgM) Monoclonal antibody (MAb) designated EB-A2for the detection of circulating Aspergillus GM have been developed, and been widely used in many countries, including USA. Although previous studies that assessed the performance characteristics of this assay reported high sensitivity and specificity, more recent studies have shown that many significant variations indeed come out by using this method. The causes of this variability are multifactorial and, in large part, cannot be explained because there is insufficient understanding of the kinetics of GM in vivo. Besides GM,(1,3)-(3-D glucan (BG) antigen is another important marker for the early serodiagnosis of deep-seated fungal infections. However, BG capturing assay cannot be used for identification at the species level, because BG is a major component of the fungal cell wall, including Aspergillus, Conidia, et al. So, the identification of other antigenic markers, may contribute to the development of new antigenic capture assays.
In2005, a whole-genome shotgun approach was finished, and the complete genomic sequence of A. fumigatus strain Af293was obtained. These made designing specific primers based on sequence alignment and genemic data very conveniently, and many researchers pay their attention to develop biomolecular diagnosis of IA. Although there are still many problems of using biomolecular diagnosis in clinic, including difficulties in standardization, biomolecular diagnosis also has many advantages, such as the accurate classification of different types in the same strain, and the acquaintance of drug resistance. Therefore, the establishment of Aspergillus specific biomolecular diagnositic method can contribute to the implementation of IA early rapid diagnosis.
In this study, we managed to build antigen-capture ELISAs and biomolecular diagnosis of I A.
1. Expression of recombinant Afmplp using pichia expression system, preparation of murine anti-Afmplp MAbs, and development of Afmplp-capture ELISA for diagnosis of A. fumigatus infection
As one of the galactomannoproteins in A. fumigates, Afmp1p is a homolog of Mplp, which is a highly antigenic cell wall mannoprotein in P. marneffei and was found to be a very useful target for the serodiagnosis of P. marneffei infections. It is speculated that Afmp1p has the same potential for use in aspergillosis serodiagnosis. Previously, we developed antigen and antibody tests using E.coli expressed GST-Afmplp and guinea pig anti-GST-Afmplp PAbs. Although no false-positive results were found in serum samples from negative control groups, sensitivity of the assays were found to be relatively low in which only8of15(53%) IA patients were Afmp1p antigen test positive. To achieve higher sensitivity and to lower the inter-and intra-laboratory variations introduced during the collection of guinea pig anti serum, MAb-based immunodiagnostic assays was proposed. A GST-Afmplp-capture ELISA using murine MAbs was developed with sensitivity of0.1ng/ml, however, it failed to capture natural Afmplp from infected sera. The failure to detect natural form of Afmplp by ELISA indicated that differences between GST-Afmplp and natural Afmp1p existed. To exclude the potential defects in prokaryotic expression systems, we selected the Pichia expression system to express Afmplp, and attempted to build an Afmplp-capture ELISA to detect A. fumigates infection.
2. Expression of recombinant Afmp4p using pichia expression system, preparation of murine anti-Afmp4p MAbs, and development of Afmp4p-capture ELISA for diagnosis of A. fumigatus infection
Like Afmplp, Afmp4p is another homolog of Mplp, and may have potential for serodiagnosis of aspergillosis. In this study, besides Afmplp, we selected the Pichia expression system to express Afmp4p too, and tried to build an Afmp4p-capture ELISA to detect A. fumigates infection.
3. Development of rDNA ITS real-time PCR for diagnosis of A. fumigatus infection
Ribosomal DNA (rDNA) codes for ribosomal RNA. The rDNA gene cluster from5' to the3' are the18S rDNA, internal transcribed spacer1(internal transcribed space, ITS1),5.8S rDNA, ITS2,28S rDNA. Among these regions,18S rDNA and ITS, consists of conserved regions and variable regions, and are good markers for biomolecular diagnosis in clinic.
In this study, we selected two pairs of PCR primer, one is pan-fungal rDNA ITS primer (ITSS:5'-GTGAATCATCGAATCTTTGAAC-3', ITSR:5'-TCCTCCGCTT ATTGATATGC-3'), the other one is pan-fungal18S rDNA primer (18SS:5'-ATTGG AGGGCAAGTCTGGTG-3',18SR:5'-CCGATCCCTAGTCGGCATAG-3'). Using the Aspergillus genomic DNA as templates, we obtained two types of different PCR products. After direct sequencing and gene sequence analysis, we seleced the better sequence for real-time PCR target, designed real-time PCR primer and probe, established a real-time PCR detecting method, but the sensitivity of this method is not high enough. In order to solve this problem, we subquently established a nested real-time PCR.
Conclusion
1. We expressed galactomannoprotein Afmplp, a potential bio-marker of A. fumigates infection. The purified rAfmplp showed as a band of proximate40kDa in western blotting, bigger than anticipation. This maybe caused by glycosylation, since Afmplp has potential O-glycosylation sites, and is expected to be a glycosylated protein. Twenty hybridoma cell lines each producing different MAbs against Afmplp were established, and were named from Afmpl-M1to Afmpl-M20. To select the optimal capturer-detector for a MAb-MAb ELISA to detect Afmp1p in A. fumigatus, the sensitivities of all possible pairs of MAbs to against rAfmp1p and native Afmp1p were determined, and Afmp1-M3and Afmp1-M6-HRP were selected as the capturing and detecting antibody for the Afmp1p capture ELISA. This ELISA would be positive even when the culture of A. fumigatus had been diluted into128-folds of its original concentration. And the limit for rAfmplp detection was approximately400pg/ml. The ELISA could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus challenged animal models. Afmp1p-capture ELISA may be useful in clinical diagnosis of aspergillosis.
2. A potential bio-marker of A. fumigates infection-galactomannoprotein, Afmp4p was expressed. The purified rAfmp4p showed as a band of proximate19kDa in western blotting. Thirteen hybridoma cell lines each producing different MAbs against Afmp4p were established, and were named from Afmp4-M1to Afmp4-M13. Afmp4-M9and Afmp4-M2-HRP were selected as the capturing and detecting antibody for the Afmp4p capture ELISA. This ELISA would be positive even when the culture of A. fumigatus had been diluted into512-folds of its original concentration, and the limit for rAfmp4p detection was approximately800pg/ml. This ELISA could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and no cross-reaction to the other Aspergillus infected rabbit sera and the sera prior infections had been observed.
3. Among these two pan-fungi PCR primers, pan-fungal18S rDNA PCR products analysis founded that all of them have the same sequence in different fungi, and pan-fungal ITS PCR products had different sequence in different fungi. These results indicated that fungal ITS region is a better target for diagnosis in species level. Though analysis of different fungal ITS sequence, we designed real-time PCR primer, the forward primer is5'-TATGGGGCTTTGTCACCTC-3',the reverse primer is5'-TCCTCCGCTTATTGATATG-3', and the Taqman MB probe is5'-CCG GCGCCAGCCGACACCCAACTTTA-3'. Using this real-time assay, we can detect A. fumigates, A. flavus and A. terrus, but the sensitivity of this method is not high enough. Sebquently, a nested real-time PCR was been built, with a pan-fungal ITS PCR augmentation before real-time PCR. This method can sepecif test the Aspergillus DNA, and other fungi, including A. nidulans, A. niger, Penicinnei marneffei, and the Condidia have negative results using this method.
In conclusion, we successfully established two antigen-capture ELISAs and a rDNA ITS detection to specifically detect A. fumigates infections in the present study. Further studies will focus on evaluating the efficacy of this Aspergillus antigen assay with clinical samples.
现阶段,烟曲霉感染的主要诊断方法包括传统诊断、基因检测、特异性抗原抗体检测等。传统的诊断方法主要有影像学、无菌腔液标本培养和组织病理学鉴定等。影像学上的新月征(air crescent)和晕轮征(halo sign),由于类似特征在念珠菌病、军团菌病、巨细胞病毒感染、Kaposi肉瘤等患者体内也可观察到,并不能作为IA确诊的依据。呼吸道分泌物培养诊断敏感性低,而血培养即使在传播性感染中,也多表现为阴性。此外,标本培养还存在培养周期长等缺点。而组织病理学检查具有创伤性,并不适用于所有的患者。因此,传统的诊断方法已难以满足IA诊断的临床需要。
IA病情发展迅速,机体并无足够产生抗体的时间,以及免疫受损患者特异性免疫应答水平低的特点,使得抗体检测易出现假阴性结果。同时,每天吸入空气中大量烟曲霉孢子,正常个体机体内所存在的烟曲霉特异抗体,又可导致假阳性结果的出现。因此,单一的特异性抗体检测在IA诊断中的应用价值一直存在争议。近年来,IA的诊断学研究热点主要集中在抗原捕获和基因检测方面。
目前被国际认可的具有诊断价值的真菌抗原主要有两种：半乳甘露聚糖抗原(Galactomannan, GM)和(1,3)-p-D葡聚糖抗原(p-D glucan, BG),这两种检测方法已被美国和欧洲许多国家批准用于侵袭性真菌感染诊断中。然而,这两种方法具有敏感性和特异性均不稳定的应有局限性。如GM检验不能区分曲霉菌与马尔尼菲青霉菌的感染,且在病情发展中很快被清除掉,导致出现假阴性结果。BG检测(G试验)没有种属特异性,不能区分曲霉与酵母菌感染,对静脉使用白蛋白或γ球蛋白患者,抗肿瘤药物如香菇菌多糖使用患者,以及粘菌素E、头孢噻肟等抗菌药物使用患者的检测,均存在出现假阳性结果的可能。寻找新的特异性抗原靶标,建立灵敏度和特异性俱佳的抗原捕获检测方法在目前仍显得尤为重要。
随着分子生物学技术的广泛应用,烟曲霉临床分离菌株Af293的全基因组测序完成,很多研究工作者把目标转向根据基因组信息设计具有种、属特异性的引物,发展简便、快速的IA诊断方法。尽管基因检测方法要成为诊断IA的常规检测方法还存在很多问题,包括方法学标准化困难,对实验场地的配置、实验操作规范性、对人员的要求高等。基因检测仍具有其它诊断方法不可比拟的优点,包括可准确分类到菌株类型,获得感染物种耐药性等相关信息。因此,建立IA患者烟曲霉特异基因检测标准化方法,有助于实现IA的早期快速精确诊断。
由此,本研究的目的主要在于,建立烟曲霉感染特异性抗原捕获ELISA检测方法,同时建立烟曲霉感染基因检测标准化诊断方法,以求最终实现烟曲霉感染的早期诊断。研究内容主要有：
1.重组Afmp1p蛋白的毕赤酵母表达,抗rAfmp1p单克隆抗体的制备以及烟曲霉感染特异性Afmplp抗原捕获ELISA方法的建立与评价
Afmplp由AFMP1基因所编码,为烟曲霉半乳甘露聚糖蛋白,由本实验室与香港大学袁国勇教授等人合作,于2001年通过生物信息学分析发现。在发现该靶标蛋白后,使用大肠杆菌表达系统表达重组GST-Afmplp融合蛋白,制备抗GST-Afmplp豚鼠血清,使用多克隆抗体(polyclonal antibody, PAb)夹心法,建立了烟曲霉感染抗原捕获ELISA检测方法。然而,该方法对烟曲霉感染IA患者检测的灵敏度仅为53%,并不能达到临床样本检测的要求。这可能是由于多克隆抗体小身所具有的缺陷造成的,包括不同批次不同动物间不可避免的血清差异。为了弥补多克隆抗体所存在的缺陷,本实验室在之前的研究中,使用GST-Afmp1p免疫小鼠,制备抗GST-Afmplp单克隆抗体(monoclonal antibody, MAb),获得了较为理想的抗原夹心捕获单克隆抗体对,可检测至0.1ng/ml的重组蛋白。遗憾的是,该方法并不能捕获天然存在的Afmp1p抗原。这可能缘于大肠杆菌表达重组GST-Afmplp蛋白由于缺乏后期修饰,与天然Afmp1p蛋白间存在构象差异,从而造成的抗原表位改变。
在本研究中,为了弥补原核表达系统所存在的无后期加工等缺陷,选择pPICZ α-B表达载体,使用野生型毕赤酵母X33菌株,表达AFMP1基因,获得了与天然蛋白更为接近的重组Afmp1p蛋白(rAfmp1p).使用rAfmp1p免疫小鼠,制备抗rAfmp1p单克隆抗体,对其进行HRP标记,鉴定亚型,并通过竞争抑制ELISA分析其识别的表位类型。在这些研究的基础上,将单克隆抗体两两配对,进行重组蛋白(10ng/μ1)夹心捕获以及感染动物血清10倍稀释液夹心捕获筛选,获得最为理想的MAb抗体对。使用所获得的最佳抗体对,对重组蛋白倍比稀释液、多种真菌培养上清稀释液以及真菌感染兔血清稀释液进行夹心检测,从而对Afmp1p抗原捕获ELISA检测方法的灵敏度与特异度进行评价。
2.重组Afmp4p蛋白的毕赤酵母表达,抗rAfmp4p MAb的制备以及烟曲霉感染特异Afmp4p抗原捕获ELISA方法的建立与评价
AFMP4基因所编码Afmp4p蛋白为Afmp1p同源蛋白,由香港大学袁国勇教授等人通过生物信息学分析发现。在Afmp1p做为烟曲霉感染临床诊断标志物的意义尚未确定之时,对Afmp4p可能的诊断意义进行分析,可增加成功建立烟曲霉感染抗原捕获ELISA检测方法的几率。因此本研究在建立Afmp1p抗原捕获ELISA检测方法的同时,同步构建pPIC9K-AFMP4重组表达载体,使用毕赤酵母表达系统表达AFMP4基因,获得重组Afmp4p蛋白(rAfmp4p),并进一步制备鼠源性抗rAfmp4p单克隆抗体,进行表位分析与亚型鉴定后,将单克隆抗体两两配对,进行重组蛋白(10ng/μ1)夹心捕获以及感染动物血清10倍稀释液夹心捕获筛选,获得最为理想的单克隆抗体对。对所获得的抗体对,使用重组蛋白倍比稀释液、多种真菌培养上清稀释液以及真菌感染兔血清稀释液夹心检测,评价方法灵敏度与特异度。
3.烟曲霉感染rDNA ITS区巢式real-time PCR检测方法的建立
核糖体DNA (ribosomal DNA, rDNA)既有保守区又有可变区,在进化速率上较为保守,作为种级以上阶元的良好标记,广泛应用于物种系统学研究中。rDNA基因簇从5’到3’端依次为18S rDNA,内转录间隔区1(internal transcribed space, ITS1),5.8S rDNA, ITS2,28S rDNA,其中,ITS Ⅰ和ITS Ⅱ常被合称为ITS区。18S rDNA序列中既有保守区又有可变区,在进化速率上较为保守,是系统发育中种级以上阶元的良好标记。ITS区由于不加入成熟核糖体,受到的选择压力较小,进化速率较快,在绝大多数的真核生物中展现出极为广泛的序列多态性,表现为种内相对一致,种间差异较明显,能真实反映属间、种间以及菌株间的碱基对差异。因此,不同物种间18S rDNA和ITS区域的序列差异常用于物种鉴别研究中。
在本研究中,选取针对真菌rDNA ITS区通用引物1对(ITSS:5'-GTGAATCAT CGAATCTTTGAAC-3', ITSR:5'-TCCTCCGCTTATTGATATGC-3'),针对真菌18S rDNA区域通用引物1对(18SS:5'-ATTGGAGGGCAAGTCTGGTG-3',18SR:5'-CCGATCCCTAGTCGGCATAG-3'),对真菌基因组DNA、烟曲霉感染兔组织(肝、肾、肺)DNA、黄曲霉感染兔组织(肝、肾、肺)DNA以及模拟真菌败血症血液标本DNA扩增,判断两种方法的检测范围。同时对曲霉基因组DNA扩增产物直接测序,经序列比较分析,选择出具有物种特异性的检测靶标序列。为了弥补通用引物扩增后,产物需进一步测序才能鉴别到物种的缺点,对不同真菌的该段序列进行比较分析,设计曲霉特异real-time PCR引物与探针,尝试建立曲霉real-time PCR检测方法。为了弥补曲霉real-time PCR检测方法灵敏度低的缺陷,进一步建立巢式real-time PCR检测。
小结
通过以上三部分的工作,本研究所获得结果主要有：
1、在毕赤酵母中成功表达并纯化rAfmp1p,获得的rAfmp1p约为40kDa,略大于预期的31.4kDa,这可能是由于蛋白糖基化造成的。使用rAfmp1p免疫小鼠,并制备属源性单克隆抗体,共获得20株抗rAfmp1p单克隆抗体,分别以Afmp1-M1至Afmp1-M20命名。亚型分析发现,除Afmp1-M1外,所有的单克隆抗体均为IgG1.表位分析发现,这20株单克隆抗体可识别6种不同的抗原表位。将这些单克隆抗体两两配对,对rAfmp1p (10ng/μ1)以及烟曲霉感染兔血清10倍稀释液双抗体夹心检测,筛选获得了特异性高,灵敏度好的单克隆抗体对,建立了烟曲霉感染Afmp1p抗原捕获ELISA检测方法。其中,包被抗体为Afmp1-M3(包被浓度为10μg/ml),捕获抗体为Afmp1-M6-HRP.该Afmplp抗原捕获ELISA检测方法对rAfmp1p检测的灵敏度为400pg/ml,可捕获到烟曲霉1640培养上清16倍稀释液中的Afmp1p抗原,可检测到烟曲霉感染24h后兔血清16倍稀释液中的Afmp1p抗原。对其它曲霉感染兔血清以及其它病原真菌1640培养上清的检测,均表现为阴性。
2、在毕赤酵母中成功表达并纯化rAfmp4p,该蛋白约为20kDa,与预期大小吻合。使用rAfmp4p免疫小鼠并制备属源性单克隆抗体,共获得13株抗rAfmp4p单克隆抗体,以Afmp4-M1至Afmp4-M13命名。亚型分析发现,所有的单克隆抗体均为IgGl。表位分析发现,这13株单克隆抗体可识别5种不同的抗原表位。将这些单克隆抗体两两配对,对rAfmp4p (10ng/μ1)以及烟曲霉感染兔血清10倍稀释液双抗体夹心检测,筛选获得了特异性高,灵敏度好的单克隆抗体对,建立了烟曲霉感染Afmp4p抗原捕获ELISA检测方法。其中,包被抗体为Afmp4-M9(包被浓度为10μg/ml),捕获抗体为Afmp4-M2-HRP。该Afmp1p抗原捕获ELISA检测法对rAfmp4p检测的灵敏度为800pg/ml,在烟曲霉1640培养上清512倍稀后,仍可捕获到释放到培养上清中的Afmp4p抗原,可检测出烟曲霉感染24h后兔血清8倍稀释液中的抗原。对其它曲霉感染兔血清以及其它病原真菌1640培养上清的检测,均表现为阴性。
3、使用真菌18S rDNA通用引物(18SS、8SR),和真菌rDNA ITS区通用引物(ITSS、ITSR),对真菌基因组DNA、烟曲霉感染兔组织(肝、肾、肺)DNA、黄曲霉感染兔组织(肝、’肾、肺)DNA以及模拟真菌败血症血液标本DNA的扩增,均表现为阳性。对扩增产物的直接测序以及Blast分析发现,扩增出的18S rDNA片段序列(504bp)为真菌共有片段,并无物种特异性,扩增出的rDNA ITS序列(299bp)具有物种特异性,不同真菌之间,以及曲霉属内不同曲霉之间均存在差异,属内物种序列相似性小于92%。使用rDNA ITS区通用引物PCR扩增与直接测序相结合,可成功对不同的真菌物种进行区分鉴别。通过不同真菌rDNA ITS区的比较分析,设计real-time PCR引物,其中上游引物为5'-TATGGGGCTTTGTCACCTC-3',下游引物为5'-TCCTCCGCTTA TTGATATG-3', Taqman特异探针为5'-CCGGCGCCAGCCGACACCCAACTTTA-3’。该方法对烟曲霉、黄曲霉以及土曲霉DNA检测表现为阳性,然而ct值高,灵敏度低。由于real-time PCR引物扩增片段区域位于ITS通用引物扩增区域内部,因此建立使用ITS通用引物第一轮扩增,real-time PCR引物第二轮扩增并荧光检测的巢式real-time PCR检测方法。该方法最适第一轮扩增循环次数为15,该方法可成功检测烟曲霉、黄曲霉以及土曲霉DNA,烟曲霉、黄曲霉感染兔组织(肝、肾、肺)DNA,以及模拟真菌败血症血液标本DNA。而对其它真菌,包括构巢曲霉、黑曲霉、马尔尼菲青霉、念珠菌、以及新生隐球菌DNA的检测,均表现为阴性。
总之,本研究成功建立了3种烟曲霉感染检测方法,其中2种为烟曲霉特异性甘露聚糖抗原捕获检测方法,1种为曲霉特异性巢式real-time PCR检测方法。遗憾的是,由于临床样本的缺乏,这些方法在临床上的应用价值,尚需进一步实验确认。
Aspergillus fumigatus is a ubiquitous filamentous fungus in the environment. On average, humans inhale hundreds of Aspergillus conidia daily. These conidia are eliminated efficiently by innate immune mechanisms in immunocompetent hosts, but could germinate and colonize in immunocompromised host. As a result of the increasing use of transplantation for end-organ disease, the use of immunosuppressive and myeloablative therapies for autoimmune and neoplastic diseases, and the human immunodeficiency virus (AIDS) pandemic, the number of immunocompromised patients is steadily expanding. In these patients, the conidia of A. fumigatus, with diameters (3to5μm) that allow them to traverse the terminal respiratory airways and reach the pulmonary alveoli, could escape the defense line and germinate in the lung, causing pulmonary aspergillosis. In most cases, the fungal pathogen will also migrate to other organs, such as myocardium, kidney, liver, spleen, soft tissue, and bone, causing invasive aspergillosis (IA)--a severe and usually fatal systematic infection. The incidence of IA can reach as high as50%in acute leukemia if the patients have a risk of Aspergillus propagation and dissemination, such as during building/ construction work. The mortality rate is often around50%. The eary diagnosis is very impotant for IA patients。
Since the signs and symptoms of IA are nonspecific, early clinical diagnosis of the infection is often difficult. The "gold standard" for diagnosis is to obtain a positive culture of A. fumigatus and to obtain histological evidence of mycelial growth in biopsy specimens. However, owing to its invasiveness, biopsy is often precluded from patients in critical conditions. A. fumigatus cultures from blood or respiratory specimens are often false-negative, especially during early stages of the disease. As an adjunctive measure to microbiological methods, antibody detection is usually performed for the diagnosis of IA, but these assays are usually insufficient owing to limits in their sensitivity and specificity. False-negative results are often come out because of the fulminant nature of the disease and the poor immunological status of the host, and false-positive results also exist in healthy individuals because of the prolong interaction with conidia. At present, most of the studies focused on the identification of antigenic markers and the DNA targets of IA.
At present, the diagnostic value of galactomannan (GM), a major component of the Aspergillus cell wall, has been identified. Platelia Aspergillus (Bio-Rad, Marnes-La-Coquette, France) assay, an immunoassays that employ a rat immunoglobulin M (IgM) Monoclonal antibody (MAb) designated EB-A2for the detection of circulating Aspergillus GM have been developed, and been widely used in many countries, including USA. Although previous studies that assessed the performance characteristics of this assay reported high sensitivity and specificity, more recent studies have shown that many significant variations indeed come out by using this method. The causes of this variability are multifactorial and, in large part, cannot be explained because there is insufficient understanding of the kinetics of GM in vivo. Besides GM,(1,3)-(3-D glucan (BG) antigen is another important marker for the early serodiagnosis of deep-seated fungal infections. However, BG capturing assay cannot be used for identification at the species level, because BG is a major component of the fungal cell wall, including Aspergillus, Conidia, et al. So, the identification of other antigenic markers, may contribute to the development of new antigenic capture assays.
In2005, a whole-genome shotgun approach was finished, and the complete genomic sequence of A. fumigatus strain Af293was obtained. These made designing specific primers based on sequence alignment and genemic data very conveniently, and many researchers pay their attention to develop biomolecular diagnosis of IA. Although there are still many problems of using biomolecular diagnosis in clinic, including difficulties in standardization, biomolecular diagnosis also has many advantages, such as the accurate classification of different types in the same strain, and the acquaintance of drug resistance. Therefore, the establishment of Aspergillus specific biomolecular diagnositic method can contribute to the implementation of IA early rapid diagnosis.
In this study, we managed to build antigen-capture ELISAs and biomolecular diagnosis of I A.
1. Expression of recombinant Afmplp using pichia expression system, preparation of murine anti-Afmplp MAbs, and development of Afmplp-capture ELISA for diagnosis of A. fumigatus infection
As one of the galactomannoproteins in A. fumigates, Afmp1p is a homolog of Mplp, which is a highly antigenic cell wall mannoprotein in P. marneffei and was found to be a very useful target for the serodiagnosis of P. marneffei infections. It is speculated that Afmp1p has the same potential for use in aspergillosis serodiagnosis. Previously, we developed antigen and antibody tests using E.coli expressed GST-Afmplp and guinea pig anti-GST-Afmplp PAbs. Although no false-positive results were found in serum samples from negative control groups, sensitivity of the assays were found to be relatively low in which only8of15(53%) IA patients were Afmp1p antigen test positive. To achieve higher sensitivity and to lower the inter-and intra-laboratory variations introduced during the collection of guinea pig anti serum, MAb-based immunodiagnostic assays was proposed. A GST-Afmplp-capture ELISA using murine MAbs was developed with sensitivity of0.1ng/ml, however, it failed to capture natural Afmplp from infected sera. The failure to detect natural form of Afmplp by ELISA indicated that differences between GST-Afmplp and natural Afmp1p existed. To exclude the potential defects in prokaryotic expression systems, we selected the Pichia expression system to express Afmplp, and attempted to build an Afmplp-capture ELISA to detect A. fumigates infection.
2. Expression of recombinant Afmp4p using pichia expression system, preparation of murine anti-Afmp4p MAbs, and development of Afmp4p-capture ELISA for diagnosis of A. fumigatus infection
Like Afmplp, Afmp4p is another homolog of Mplp, and may have potential for serodiagnosis of aspergillosis. In this study, besides Afmplp, we selected the Pichia expression system to express Afmp4p too, and tried to build an Afmp4p-capture ELISA to detect A. fumigates infection.
3. Development of rDNA ITS real-time PCR for diagnosis of A. fumigatus infection
Ribosomal DNA (rDNA) codes for ribosomal RNA. The rDNA gene cluster from5' to the3' are the18S rDNA, internal transcribed spacer1(internal transcribed space, ITS1),5.8S rDNA, ITS2,28S rDNA. Among these regions,18S rDNA and ITS, consists of conserved regions and variable regions, and are good markers for biomolecular diagnosis in clinic.
In this study, we selected two pairs of PCR primer, one is pan-fungal rDNA ITS primer (ITSS:5'-GTGAATCATCGAATCTTTGAAC-3', ITSR:5'-TCCTCCGCTT ATTGATATGC-3'), the other one is pan-fungal18S rDNA primer (18SS:5'-ATTGG AGGGCAAGTCTGGTG-3',18SR:5'-CCGATCCCTAGTCGGCATAG-3'). Using the Aspergillus genomic DNA as templates, we obtained two types of different PCR products. After direct sequencing and gene sequence analysis, we seleced the better sequence for real-time PCR target, designed real-time PCR primer and probe, established a real-time PCR detecting method, but the sensitivity of this method is not high enough. In order to solve this problem, we subquently established a nested real-time PCR.
Conclusion
1. We expressed galactomannoprotein Afmplp, a potential bio-marker of A. fumigates infection. The purified rAfmplp showed as a band of proximate40kDa in western blotting, bigger than anticipation. This maybe caused by glycosylation, since Afmplp has potential O-glycosylation sites, and is expected to be a glycosylated protein. Twenty hybridoma cell lines each producing different MAbs against Afmplp were established, and were named from Afmpl-M1to Afmpl-M20. To select the optimal capturer-detector for a MAb-MAb ELISA to detect Afmp1p in A. fumigatus, the sensitivities of all possible pairs of MAbs to against rAfmp1p and native Afmp1p were determined, and Afmp1-M3and Afmp1-M6-HRP were selected as the capturing and detecting antibody for the Afmp1p capture ELISA. This ELISA would be positive even when the culture of A. fumigatus had been diluted into128-folds of its original concentration. And the limit for rAfmplp detection was approximately400pg/ml. The ELISA could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus challenged animal models. Afmp1p-capture ELISA may be useful in clinical diagnosis of aspergillosis.
2. A potential bio-marker of A. fumigates infection-galactomannoprotein, Afmp4p was expressed. The purified rAfmp4p showed as a band of proximate19kDa in western blotting. Thirteen hybridoma cell lines each producing different MAbs against Afmp4p were established, and were named from Afmp4-M1to Afmp4-M13. Afmp4-M9and Afmp4-M2-HRP were selected as the capturing and detecting antibody for the Afmp4p capture ELISA. This ELISA would be positive even when the culture of A. fumigatus had been diluted into512-folds of its original concentration, and the limit for rAfmp4p detection was approximately800pg/ml. This ELISA could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and no cross-reaction to the other Aspergillus infected rabbit sera and the sera prior infections had been observed.
3. Among these two pan-fungi PCR primers, pan-fungal18S rDNA PCR products analysis founded that all of them have the same sequence in different fungi, and pan-fungal ITS PCR products had different sequence in different fungi. These results indicated that fungal ITS region is a better target for diagnosis in species level. Though analysis of different fungal ITS sequence, we designed real-time PCR primer, the forward primer is5'-TATGGGGCTTTGTCACCTC-3',the reverse primer is5'-TCCTCCGCTTATTGATATG-3', and the Taqman MB probe is5'-CCG GCGCCAGCCGACACCCAACTTTA-3'. Using this real-time assay, we can detect A. fumigates, A. flavus and A. terrus, but the sensitivity of this method is not high enough. Sebquently, a nested real-time PCR was been built, with a pan-fungal ITS PCR augmentation before real-time PCR. This method can sepecif test the Aspergillus DNA, and other fungi, including A. nidulans, A. niger, Penicinnei marneffei, and the Condidia have negative results using this method.
In conclusion, we successfully established two antigen-capture ELISAs and a rDNA ITS detection to specifically detect A. fumigates infections in the present study. Further studies will focus on evaluating the efficacy of this Aspergillus antigen assay with clinical samples.
引文
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