【作者】 史利宁；

【导师】 邵世和；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2012， 博士

【摘要】 背景近年来,侵袭性曲霉病(invasive aspergillosis, IA)己成为中性粒细胞缺乏症患者重要的死亡原因之一,而且在非中性粒细胞缺乏症患者如ICU危重患者甚至免疫功能正常的患者,IA的发病率亦逐年上升。由于缺乏特异性临床症状和体征,传统的诊断方法如组织病理学检查和真菌培养在感染早期阶段缺乏敏感性,因此IA的诊断仍困难,常常到疾病晚期才能诊断或死亡后由尸解获得。目前诊断IA最有价值的实验是检测曲霉细胞壁半乳甘露聚糖(galactomannan, GM)的GM实验。然而,GM实验的敏感性和特异性尚不能满足临床需求。因此,迫切需要研究发现在感染患者血液或其他体液中出现的真菌抗原,以筛选更敏感、更准确的疾病标志物用于IA的早期诊断。在前期研究中,我们发现确诊IA患者血清中常存在高滴度抗曲霉抗体,这一发现促使我们进一步深入研究能用于诊断IA的新的诊断标志物。目的应用免疫蛋白质组学方法筛选鉴定烟曲霉免疫优势抗原；应用分子克隆和原核表达技术,获得烟曲霉新的免疫优势抗原硫氧还蛋白还原酶GliT (TR)的全长重组蛋白；以重组烟曲霉硫氧还蛋白还原酶GliT抗原为包被抗原,建立测定人血清中抗烟曲霉硫氧还蛋白还原酶GliT抗体的ELISA法；用IA家兔模型,考察实验动物产生特异性anti-TR抗体应答的情况；通过检测临床血样本评估该法在侵袭性曲霉病诊治中的应用价值。方法TCA/丙酮沉淀法制备烟曲霉分泌蛋白及菌体蛋白质,用确诊IA患者血清与烟曲霉分泌蛋白及菌体蛋白质进行免疫印迹,筛选免疫反应最强的蛋白质。筛选出的免疫反应最强的烟曲霉分泌蛋白质进一步用二维电泳来分离,并用混合确诊患者血清进行免疫印迹。从2-DE胶上切下有免疫反应的蛋白质点进行胶内酶切以及基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析获得肽质量指纹图谱,然后利用Mascot查询软件搜索NCBI数据库鉴定蛋白质。对免疫反应最强的优势抗原烟曲霉TR进行生物信息学分析。用SignalP软件预测该蛋白质是否有信号肽,用WoLF PSORT预测其亚细胞定位,并用BLAST程序进行同源性分析。用RT-PCR,从烟曲霉总RNA中扩增出TR cDNA片段,克隆至pMD18-T载体,并转化E.coli JM109。经序列分析证实后,提取重组克隆质粒双酶切获得目的基因,与表达载体pET28a(+)连接,转化E.coli BL21 (DE3),筛选重组表达质粒。用异丙基硫代-p-D-半乳糖苷(IPTG)诱导重组融合蛋白表达,用SDS- PAGE及免疫印迹法分析重组蛋白,亲和层析柱纯化重组蛋白。新西兰白兔注射可的松造模,静脉注射烟曲霉分生孢子,建立IA动物模型。以重组烟曲霉TR为包被抗原,建立检测；anti-TR抗体的间接ELISA法,并观察IA家兔模型血清中anti-TR抗体产生的情况。收集经组织病理学检查和/或真菌培养证实的各类IA患者血清,以健康体检者、无IA但血培养为其他真菌及细菌的患者血清为对照,用ELISA法测定血清中的anti-TR水平,确定cut off值并对方法的敏感性和特异性进行考察。结果(1)烟曲霉分泌蛋白质的免疫反应性在反应强度及反应数量上均优于菌体蛋白质。在4种不同培养基的分泌蛋白质中,用YEPG培养烟曲霉14d的分泌蛋白质的免疫反应性最强,有免疫反应的蛋白质条带数量最多,拟用于免疫蛋白质组学分析,进一步筛选特异性烟曲霉抗原。(2)烟曲霉分泌蛋白质的免疫蛋白质组学分析结果显示,确诊IA患者血清与分泌蛋白质中多种抗原反应强烈。40个蛋白质点被抗体阳性IA患者血清识别。对40个蛋白质点质谱分析,成功鉴定了39个免疫反应阳性的蛋白质点,代表17种蛋白质。多数鉴定蛋白质为参与碳水化合物、脂肪酸、氨基酸和能量代谢的代谢酶。在鉴定出的17种蛋白质中,己知有7种蛋白质为曲霉或其他真菌抗原,有10种为新发现的有免疫原性的蛋白质,如延胡索酰乙酰乙酸水解酶FahA、醛脱氢酶AldA、芳香基氨基转移酶Aro8、G-蛋白复合物p亚基CpcB、肌动蛋白细胞骨架蛋白(VIP1)、phytanoyl-CoA二氧化酶家族、尿酸氧化酶UaZ、3-羟丁酰辅酶A脱氢酶、蛋白酶体组分Pre8和假想蛋白。有7种免疫原性蛋白显示有多个蛋白质点。令我们感兴趣的一个免疫反应最强的蛋白质,鉴定为硫氧还蛋白还原酶GliT (TR)。(3)用SignalP软件预测结果显示TR带有信号肽(signalP probability,0.808); WoLF PSORT软件预测结果显示TR为胞外蛋白质(Query Protein WoLFPSORT prediction: extr,12.0; cyto,6.5; cyto_nucl,4.0; mito,3.0; pero,2.0);用BLAST程序进行同源性分析显示,TR与人源蛋白质没有同源性,且与其他真菌同源性低,如与白念珠菌蛋白质的同源性为25%,热带念珠菌25%,光滑念珠菌24%,季也蒙念珠菌27%,酿酒酵母菌24%,马尔尼菲青霉27%。因此,是一个理想的诊断标志物。(4)成功构建了含烟曲霉TR全长基因的重组表达质粒,重组质粒中目的片段测序结果显示与GenBank公布的烟曲霉TR全长基因的编码序列完全一致。重组表达工程菌经IPTG诱导,超声破碎后裂解上清经SDS-PAGE在分子质量(Mr)约36000处出现目的蛋白质条带,经质谱鉴定为烟曲霉TR,序列覆盖度37%。。多数重组蛋白质为可溶性蛋白；经Talon金属亲和层析柱纯化后,SDS-PAGE见单一重组蛋白质条带,纯度达91.1%。纯化后的TR蛋白经电泳分离后,分别与抗His标签单抗、确诊IA患者血清及健康人混合血清进行免疫印迹。结果显示：重组蛋白带有His标签并具有免疫反应性,可与IA患者血清特异性结合,而与健康人混合血清无反应。(5)用家兔IA模型评估对烟曲霉TR的抗体应答情况,结果显示家兔在烟曲霉感染后7天即出现anti-TR抗体,且抗体滴度快速上升。提示anti-TR抗体在IA早期阶段即产生,可以作为诊断IA的可靠标志物。(6)以重组TR抗原为包被抗原建立的检测(?)anti-TR抗体ELISA法精密度良好,批内CV%=8.57%,批间CV%=12.17%。anti-TR抗体检测诊断非粒缺患者IA的敏感性和特异性达80.9%和96%,明显优于GM实验(p<0.01)。35.9%(15/42)非粒缺IA患者anti-TR阳性而GM始终阴性。在入院首次血样检测中,69%(29/42)非粒缺IA患者anti-TR抗体阳性。联合检测anti-TR和GM,可使敏感性提高至88.1%。结论我们用免疫蛋白组学方法鉴定了IA疾病过程中表达的17种抗原,其中10种蛋白质首次报道有抗原性。我们克隆表达了其中一个免疫反应最强的抗原,即烟曲霉硫氧还蛋白还原酶GliT,并建立了检测anti-TR抗体的ELISA法。我们首次评估了该ELISA法诊断IA的敏感性和特异性,并证实anti-TR抗体是非粒缺患者早期诊断IA的有用标志物。更多还原

【Abstract】 Background:In recent decades, invasive aspergillosis (IA) has emerged as an important cause of morbidity and mortality in patients with prolonged neutropenia. Moreover, several reports have recently described a rising incidence of IA in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is still difficult because signs and symptoms are non-specific. The conventional diagnostic methods, such as tissue examination and microbial cultivation, may lack sensitivity in the first stages of infection in critically ill patients. As a result, the diagnosis of IA is often established after a long delay or following autopsy. Currently, the most valuable method used in the diagnosis of IA is GM assay. Galactomannan (GM) is present in the cell walls of most Aspergillus species. However, the sensitivity and specificity of GM testing can not meet clinical needs. Therefore, more prompt and accurate disease markers for early diagnosis are needed, which requires a thorough knowledge of fungal antigens detected in the serum or other body fluids of infected patients. We have recently observed that high levels of antibody against A. fumigatus are often present in the sera of proven IA patients. This finding prompted us to discover the potential novel biomarkers for the diagnosis of IA.Objective:To screen and identify Aspergillus fumigatus immunodominant antigens using immunoproteomics. To obtain the full-length recombinant protein of thioredoxin reductase GliT (TR), the novel immunodominant antigen identified from A. fumigatus. To establish an ELISA-based method for detecting anti-TR antibodies using the recombinant TR as the coating antigen. To investigate the antibody response to TR by a rabbit model of invasive aspergillosis. To evaluate the clinical significane of such anti-TR antibody detection method in diagnosis and therapy monitoring of IA by clinical serum samples.Methods:The secreted proteins and mycelial proteins of A. fumigatus prepared by TCA/acetone method were used for immunoblot assay with the sera of proven IA patients in order to select strongest immunoreactive proteins. The secretory proteins of A. fumigatus, which showed the high immunoreactivity, were separated by 2-DE, and probed with pooled sera of patients with proven IA. The sepecific immunoreactive proteins spots were excised from the 2-DE gels for tryptic in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF-MS analysis were searehed against the NCBI database using Mascot software. One of the proteins, thioredoxin reductase GliT (TR), which showed the best immunoactivity, was further analyzed by bioinformatics. The signal peptide and the probability of TR were predicted using SignalP software. Another subcellular localization prediction tool, WoLF PSORT, was used to analyze the amino acid sequences of proteins for prediction of cellular localization. Homology analysis was performed using the BLAST program. From total RNA of A. fumigatus, cDNA was generated using reverse transcription PCR. The amplified fragment was cloned into pMD18-T vector and sequenced. The recombinant plasmid pMD18-T/TR was digested by the restriction enzymes, and the target fragment was inserted into pET-28a(+) vector. Then, it was used to transform E.coli BL21(DE3) and induced expression by IPTG. The recombinant TR protein was analyzed by SDS-PAGE and western blot, and purifed by TALON Metal Affinity Resins.New Zealand white rabbits were immunosuppressed by intramuscular injection of cortisone acetate. The rabbits were infected intravenously withAspergillus conidia. An indirect ELISA detect the anti-TR antibody levels in the sera was established using the recombinant TR as coating antigen. The rabbit IA model was used to further evaluate the antibody response to TR. Sera from patients with culture- and/or histology-documented IA were obatined. Control sera were obtained from healthy individuals and non-neutropenic patients with culture-documented candidemia and bacteremia. The anti-TR antibody level was detected by ELISA. The sensitivity and specificity of the test were investigated and cut-off value was determined.Results:(1) The immunoreactivity of secretory proteins was higher than that of mycelial proteins. Among 4 kinds of media, proteins secreted by A. fumigatus during growth in YEPG for 14 days showed the highest immunoreactivity and the most immunoreactive bands, which could be further analyzed by immunoproteomic approach to screen specific anitgens of A. fumigatus.(2) The 2-DE and Western blot analyses of the filtrate proteins showed that IA patient sera reacted strongly with many antigens of secretory proteins. A total of 40 distinct immunoreactive spots were identified. The 39 successfully identified spots corresponded to 17 individual proteins. Most of these proteins are metabolic enzymes that are involved in carbohydrate, fatty acid, amino acid, and energy metabolism. Seven of these proteins have been reported as antigens of Aspergillus and other fungi, and others have not been described as antigens before, such as fumarylacetoacetate hydrolase FahA, aldehyde dehydrogenase AldA, aromatic aminotransferase Aro8, G-protein comlpex beta subunit CpcB, actin cytoskeleton protein (VIP1), phytanoyl-CoA dioxygenase family, urate oxydase UaZ,3-hydroxybutyryl-CoA dehydrogenase, proteasome component Pre8, putative and hypothetical protein. Seven proteins occurred in multiple spots. One protein of interest, which showed the best immunoreactivity, was identified as TR.(3) TR was predicted as a secretory protein with the presence of signal sequences with good predictive value (signalP probability,0.808). The protein localization of TR was predicted using WoLF PSORT, and the result also indicated that this protein might be an extracellular protein (Query Protein WoLFPSORT prediction:extr,12.0; cyto,6.5; cyto_nucl,4.0; mito,3.0; pero,2.0). This protein was BLAST-searched for sequence homology with human proteins and other fungi using the BLAST program. The results indicated that TR of A. fumigatus had no matches with human proteins. Furthermore, TR of A. fumigatus had low homology with other fungi, such as Candida albicans (25%), C. tropicalis (25%), C. glabrata (24%), C. guilliermondii (27%), C. dubliniensis (23%), Saccharomyces cerevisiae (24%), Cryptococcus neoformans (28%), and Penicillium marneffei (27%). These results suggested that the TR of A. fumigatus could be developed as a biomarker for the diagnosis of IA.(4) The full-length TR gene were cloned into the pET-28a (+) expression vector. The TR sequence was 100% identical to the gene of A. fumigatus published in GenBank database. After induction by isopropyl-β-D-thiogalactoside (IPTG), the recombinant 6-His-tagged TR was expressed, and a novel protein band corresponding to 36 kDa was detected by SDS-PAGE. Protein identity was unambiguously confirmed by MALDI-TOF MS, whereas following tryptic digestion proteins were identified yielding 37% sequence coverage. Most of the recombinant proteins were soluble. After purification using a TALON metal affinity resin, the protein purity was approximately 91%. Western blot showed that the recombinant proteins could be recognized by the sera from all six patients with proven IA。(5) A rabbit IA model was used to further evaluate the antibody response to TR. An increased amount of antibody was detectable in the serum 7 days after infection. The antibody level showed a constant rise around the time of clinical evidence of IA. This would imply that anti-TR antibodies are probably produced early in the development of IA and indicates the utility of anti-TR antibodies as a reliable marker for IA.(6) An ELISA to detect the anti-TR antibody levels was set up using the recombinant TR as coating antigen. The intra-assay variation and inter-assay variation of the ELISA were 8.57% and 12.17%, respectively. The anti-TR antibody detection had a sensitivity of 80.9% and a specificity of 96%. Overall, the sensitivity of the anti-TR antibody test in diagnosing IA was better than detection of GM (52.3%) in serum (p< 0.01). The anti-TR antibody in the first serum sample was positive in 15 of the 42 cases (35.9%, 15/42), whereas the serum GM remained negative. The first serum sample from 29 of 42 antibody-positive patients (69%) showed a positive result. By combining the GM assay with the anti-TR ELISA, the diagnostic sensitivity among non-neutropenic patients increased to 88.1%.Conclusions:Using immunoproteomics approach, a total of 17 proteins of A. fumigatus were identified as antigens espressed in IA. Ten of the proteins have not been reported as antigens of Aspergillus and/or other fungi. One protein of interest, which showed the best immunoreactivity, was identified as TR. The recombinant TR was cloned and expressed, then a recombinant TR-based ELISA to detect the anti-TR antibody levels was established. The test’s performance was assessed by measuring the sensitivity and specificity in discriminating IA patients from non-IA patients. The results indicate that the anti-TR antibody is a useful marker in establishing the diagnosis of IA in non-neutropenic patients.更多还原