六种虫媒病毒蛋白芯片检测方法的建立

【作者】 林方；

【导师】 祝庆余； 杨银辉；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2012， 博士

【摘要】 虫媒病毒（Arthropod viruses）是一大类通过嗜血节肢动物传播的病毒，通常引起人类发热性和脑炎样疾病，流行病学呈现明显的季节性与地域性，多爆发于蚊虫、蜱类等大量孽生的夏秋季节，严重危害人类健康与公共安全。由于一些虫媒病毒还能通过气溶胶途径传播，也是重要的病毒性生物战剂。虫媒病毒包括披膜病毒科、布亚病毒科、黄病毒科在内的多种病毒，其中以黄病毒属病毒感染的危害最大，在我国境内主要流行的虫媒病毒病包括流行性乙型脑炎、森林脑炎、登革热、新疆出血热，多是由黄病毒属病毒感染所致的传染病。虫媒病毒的致病机制尚不清楚，目前亦无有效的疫苗和抗病毒药物。早期、快速鉴定病原体是有效预防和控制虫媒病毒病的基础。由于虫媒病毒感染的临床表现与流行病学特点相似，因此，准确检测与鉴定病原体显得尤为重要。目前，对于虫媒病毒抗体的临床检测技术仍以传统的免疫荧光（IFA）和ELISA检测为主，一次反应只能检测一种病原体。本研究以黄病毒属的流行性乙型脑炎病毒(JEV)、森林脑炎病毒(TBEV)、登革病毒(DENV)、西尼罗病毒(WNV)和甲病毒属的西部马脑炎病毒(WEEV)、东部马脑炎病毒(EEEV)为研究对象，建立能同时检测六种虫媒病毒抗体的蛋白芯片检测方法，为虫媒病毒病的临床诊断提供新的手段。检测原理是将病毒特异性诊断抗原作为捕获抗原点样制备蛋白芯片，利用抗原抗体反应的原理检测血清中的病毒特异性抗体，再通过与荧光标记的二抗反应进行结果的分析与判定。目前，国内外尚未见到蛋白芯片同时检测多种虫媒病毒抗体的报道。本课题的主要研究结果如下：1、虫媒病毒候选诊断抗原的表达、纯化及鉴定本课题选择含有诱导中和抗体的抗原表位的重组抗原作为蛋白芯片检测技术的候选诊断抗原。研究表明，黄病毒属病毒的EDⅢ蛋白只包含诱导中和抗体的型与亚型特异性抗原表位，不含有诱导产生交叉反应抗体的抗原表位，NS1蛋白可诱导中和抗体，是目前疫苗研究与病毒免疫诊断的研究热点，PrM蛋白和NS3蛋白在黄病毒属中保守稳定，具有良好免疫原性，这两个蛋白均有作为病毒诊断抗原的报道。甲病毒属的E1蛋白含有中和性抗原决定簇，至少有4个非重叠的抗原表位，具有良好的免疫原性，是甲病毒属病毒免疫学检测的重要抗原之一。依据国内外对这两类病毒诊断抗原的研究进展，并结合我国病毒流行株的特点，本课题使用DNAstar与Primer5软件进行引物设计并添加适当的酶切位点，共设计了针对TBEV、JEV、DENV1、DENV2、DENV3、DENV4、YFV、WEE、EEE、WNV的17对PCR扩增引物。通过构建并鉴定重组表达质粒，在大肠杆菌中共表达了17个重组抗原，并采用镍离子亲和层析的方法进行了抗原的纯化。另外，利用灭活病毒免疫大白兔制备动物免疫血清，在完成其效价及特异性鉴定的基础上，采用ELISA方法通过动物免疫血清鉴定所表达的重组抗原的检测特异性，共获得12个具有良好特异性的诊断抗原，为进一步建立虫媒病毒蛋白芯片检测方法奠定了基础。2、蛋白芯片检测方法的建立及条件优化采用SpotArrayTM24点样仪将纯化后的17个候选诊断抗原作为捕获抗原点制于晶芯高分子三维基片上制备蛋白芯片，以兔IgG作为阳性对照，以含15%甘油的PBS为阴性对照，每个样本重复3点，点样后，玻片室温下自然风干4h。首先采用兔免疫血清与蛋白芯片进行反应，筛选可用于蛋白芯片检测方法的特异性良好的诊断抗原。通过分析，共筛选出12个特异性较好的重组抗原，与ELISA法筛选的结果一致，表明将不同病毒的诊断抗原在一张芯片上反应并不影响彼此的检测特异性。在此基础上，进行蛋白芯片检测条件的优化，优化结果显示，抗原点样浓度在0.125～0.6mg/ml之间可获得良好的检测特异性，血清检测范围可达到1：100～1：1000。蛋白芯片的最佳反应条件芯片封闭时间为2h、Cy3标记的羊抗兔IgG使用效价为1：1000、第二抗体反应时间为45min，蛋白样本稀释液中甘油的浓度为15%。在条件优化之后，再次用兔免疫血清进行蛋白芯片的验证，证明用优化后的反应条件及筛选到的12个诊断抗原可获得良好的检测特异性。3.蛋白芯片检测方法的考核与验证建立虫媒病毒蛋白芯片检测方法的最终目的是将其应用于虫媒病毒感染患者的临床诊断，为虫媒传染病的诊断和治疗提供重要的病原学依据。因此，在建立蛋白芯片检测方法之后，采用临床血清标本对芯片的实际检测效果进行了考核与验证。共收集了33份疑似森林脑炎病毒感染血清、8份疑似乙型脑炎病毒感染血清、22份疑似登革病毒感染血清，以及20份正常人血清作为阴性对照。首先，采用以灭活全病毒作为抗原的IFA方法对样本进行了检测，然后用筛选出的12个诊断抗原作为包被或捕获抗原，采用ELISA方法和优化后的蛋白芯片方法进行了平行检测，以比较其检测效果。在33份疑似森林脑炎病毒感染血清中，IFA方法检测结果为21份IgG抗体阳性，23份IgM抗体阳性；在重组抗原的平行检测中，ELISA和蛋白芯片的检测结果均为16份IgG抗体阳性、17份IgM抗体阳性，检测符合率达到100%；对8份疑似乙型脑炎病毒感染血清的检测中，IFA检测到8份IgG抗体阳性，6份IgM抗体阳性，ELISA法检测到4份IgG抗体阳性、5份IgM抗体阳性，而蛋白芯片法检测到5份IgG抗体阳性和5份IgM抗体阳性，检出率稍高于ELISA法；对22份疑似登革病毒感染血清的检测结果为：ELISA检测13份IgG抗体阳性，15份IgM抗体阳性，而蛋白芯片法检出14份IgG抗体阳性，15份IgM抗体阳性，检出率稍高于ELISA法。ELISA与蛋白芯片方法检测出的阳性血清标本编号一致，而且均在IFA检测的阳性标本范围中。经Kappa检验后,证明蛋白芯片与ELISA具有良好的一致性。正常人血清样本检测结果均为阴性。蛋白芯片法在检测出DENV抗体阳性的基础上，可以同时对标本进行血清学分型。以重组抗原为捕获抗原的蛋白芯片与ELISA法的阳性检出率均低于以全病毒为抗原的IFA方法。为提高蛋白芯片方法的敏感性，以TBEV为例，用EDⅢ与NS1抗原组合作为诊断抗原进行检测，共检出18份IgG阳性血清和18份TBEV IgM阳性血清，比单一TBE-EDⅢ抗原多检出IgG抗体阳性标本2份，IgM阳性标本1份，证明应用组合抗原可提高抗体阳性标本的检出率。通过对诊断抗原的鉴定与筛选以及对蛋白芯片检测条件的优化，本研究建立的6种虫媒病毒蛋白芯片检测方法获得了较好的检测特异性与敏感性，对临床血清标本的检测特异性达到100%，敏感性稍高于传统的ELISA法。进一步的研究结果表明，采用组合的诊断抗原，可以提高蛋白芯片的阳性检出率。与传统的IFA和ELISA抗体检测方法相比，蛋白芯片除具有检测通量高的优势外，还可以节约检测成本、缩短检测时间以及减少对抗原和临床样本的需求量。本课题首次建立了可用于同时检测6种不同虫媒病毒抗体的蛋白芯片方法，并可对DENV的不同血清型进行分型，为虫媒病毒病的临床诊断提供了新的技术手段。更多还原

【Abstract】 Arboviruses (arthropod-borne viruses) are viruses transmitted by hematophagous arthropodvectors and susceptible vertebrate hosts. Arboviruses cause infections in humans, which are mostlyasymptomatic but can extend from mild fever to severe encephalitis. The epidemiology ofarboviruses typically presents as seasonal and locally distribution. Arboviruses emerge maily insummer and autumn which is positively correlates with the active season of the tick and mosquitovectors, and severely threat human health and public security. As some of the arboviruses cantransmit by aerosols and regarded as important viral bio-warfare agents. Arboviruses include manyviruses within the family Togaviridae, Bunyaviridae and Flaviviridae. Among them, Flavivirusinfection is the most dangerous. Japanese encephalitis virus (JEV), dengue virus (DENV),Crimean-Congo hemorrhagic fever virus (CCHFV)(also known as Xinjiang hemorrhagic fevervirus, XHFV), and tick-borne encephalitis virus (TBEV) are the four principal arboviruses of publichealth importance in China’s mainland at present. The pathogenesis of these viruses is still unclearand there are no effective vaccines or antivirus medicines so far. The specific early diagnosis of theinfection is critical for the effective prevention and therapy.Because of the similar clinical symptoms and epidemiology, the accurate detection andidentification of pathogens is of critically importance. Most frequently used diagnosis for antibodiespresently are IFA and ELISA which can detect one target in one reaction. The aim of our presentstudy was to establish a protein-chip platform to detect antibodies against JEV, TBEV, DENV,WNV, WEEV and EEEV simutaniously. This will provide a new way for clinical diagnosis ofarboviruses infection. The principal of the platform is to select the specific diagnostic antigens ascapture antigen to spot on chip to react with antiviral antibodies, and then to combine with afluorescence labeled second antibody for analysis and determination of the results. So far, noprotein array for detection of anti-arbovirus antibodies was reported.The main results of our study are summarized as following:1. The expression, purification and identification of arbovirus potential diagnostic antigensWe selected recombine antigens include epitopes to induce neutralizing antibody as ourpotential diagnose antigens for the protein array. This is based on the previous findings that-EDⅢprotein of Flavivirus consists of specific type and subtype epitopes to induce neutralizing antibodywithout epitopes for inducing cross-react antibodies. Structural protein PrM, nonstructural proteinNS1and NS3can also induce neutralizing antibody and have been employed as diagnostic antigens.It is also reported that E1protein of Alphavirus includes at least four non-lapped epitopes to induce neutralizing antibody and have widely been used as diagnostic antigens. Combined with the specialcharacteristics of viral strains epidemic in our country, we used DNAstar and Primer5softwares todesign specific PCR primers and added the suitable enzyme digest cites. Totally,17pair of primerswere designed for amplification of genes of TBEV、JEV、DENV1、DENV2、DENV3、DENV4、YFV、WEE、EEE and WNV. Recombinant expression plasmids were constructed and verified byRT-PCR and sequencing. Seventeen combined antigens were expressed by E.coli and purified. Inaddition, animal immune serum were prepared by inoculate rabbits with inactive whole virus andtheir titers and specificity were confirmed. The specificity of expressed antigens was identified byELISA through react with animal immune serum. Twelve diagnostic antigens were selected withgood specificity. These diagnostic antigens provide good materials for establishment of proteinarray.2. The establishment of protein array and the optimization of reaction parametersSeventeen expressed potential diagnostic antigens were spotted on a three-dimensional chip toconstruct protein-array. Rabbit IgG was used and positive control and PBS contained20%glycerinewas used as negative control. Each antigen was spotted three times and air-dried at roomtemperature. Firstly, rabbit immune serum was used to react with the spotted protein array to selectthe diagnostic antigens for capture antigens. After reaction and analyse, twelve diagnostic antigenswere selected with good specificity, the same as selected with ELISA method. This indicated thatcombined different antigens on one chip did not affect their individual specificity. Then, thereaction parameters were optimized. The results showed that when concentration of antigens werewithin the scale of0.125～0.6mg/ml could obtain good specificity. The detected antibodies rangedfrom1：100to1:1000. The optimized parameters were as following:2hrs of inculbation time,1:1000of the dilution of second antibody,45min of incubating period of second antibody, theconcentration of glycerine was15%of the protein dilution buffer. After the opitimization, theprotein array was verified with the immune serum again. All12antigens showed good specificityon the array.3. The evaluation and validation of the protein arrayThe purpose of this study is to apply the protein array to clinical diagnosis of arbovirusinfected patients, and to provide an etiological evidence for the treatment of arbovirus infecteddiseases. Therefore, after the establishment and optimization of the protein array, we used clinicalpatient serum for the evaluation and validation of the method. Totally,33suspected TBEV infectedsera,8suspected JEV infected sera and22suspected DENV infected sera were collected and another20sera from healthy voluteers were used as negative controls. Firstly, IFA coated withwhole virus was employed to detect the suspected serum. Then identical ELISA and protein arrayused the same antigens were carried out in parrellel for comparison.The results showed among the33suspected TBEV infected serum,23were TBEV IgG positive and23were IgM positive detectedwith IFA, while identical ELISA and protein array all tested16IgG positive and17IgM positive,the correlation of ELISA and protein array was100%. Among the8suspected JEV infected serum,8were JEV IgG positive and6were IgM positive detected with IFA, while the traditional ELISAtested4IgG positive and5IgM positive, and protein array detected5IgG positive and5IgMpositive. Among the22suspected DENV infected serum,13were DENV IgG positive and15wereIgM positive detected with ELISA, and14IgG positive and15IgM positive detected by proteinarray, the sensitivity of protein array was slightly higher than the ELISA. All healthy serum gavenegative results. Protein array has very good correlation with the ELISA after Kappa statistical test.Protein array and the identical ELISA used the recombinant antigens as capture antigen had lowerpositive numbers compared with the IFA used whole virus as capture antigen. In order to increasethe sensitivity of the protein array, we mixed EDⅢ and NS1antigens as capture antigen fordetecting of TBEV antibodies.20TBEV IgM positive sera were identified. Combined antigencould increase the sensitivity of the protein array method.After screening and optimization of the potential antigens and the reaction parameters,relatively good specificity and sensitivity were obtained. The specificity of the protein array fordetecting of clinical serum was100%and the sensitivity was as well as the traditional ELISA.Additional study indicated that by combining the antigens, the sensitivity of the protein array couldbe increased.This study reported a protein array for detection of antibodies against six arboviruses for thefirst time. The protein array can detect six viruses as well as subtype of four DENV subtypes. Itprovides a new method for diagnosis of the six arboviruses infected diseases.更多还原