Ⅲ型登革病毒NS1蛋白特异性单克隆抗体的制备、鉴定及方法学建立

发布时间：2018-06-08 10:49

本文选题：III型登革病毒 + 非结构蛋白1　；参考：《南方医科大学》2008年硕士论文

【摘要】： 登革病毒(DV)属于黄病毒科黄病毒属,通过埃及伊蚊和白蚊伊蚊传播,登革病毒包括4种血清型(DV1～4),任何型别的登革病毒感染均可引起一系列的临床症状,表现为隐性感染、发热、登革热(DF)、或更为严重的登革出血热(DHF)和登革休克综合征(DSS)。登革病毒主要流行于热带和亚热带地区,近100多个国家、25亿人口受到威胁,在登革疫区中,多见4型登革病毒交替流行,更增加了不同型别反复感染的危险性。登革热已成为一种严重危害人类健康的蚊媒病毒传染病。初次感染登革病毒所获得的免疫力对于同型病毒的再次感染可产生终身的免疫保护作用,但是对于其它型别登革病毒的再次感染仅能产生部分而且短暂的免疫保护作用。由于存在抗体依赖的感染增强作用,异型DV再次感染是发生DHF/DSS的主要危险因素。目前具有保护性的登革病毒疫苗尚未研制成功,临床上对于登革病毒的感染亦未有特异性的治疗措施。但研究表明及早的临床处理可以减少DHF的发病率和致死率。由于多数登革病毒感染者早期缺乏特异的临床表现,DV感染确诊依赖于实验室诊断手段,从血液或组织中分离到病毒,检测病毒抗原或RNA或者检测血清特异性的抗体。目前,登革病毒的实验室诊断方法主要包括病毒分离、血清学检测和核酸检测。由于4种登革病毒的血清型之间,以及与其它黄病毒成员间存在共同抗原表位,用传统的血清学方法如血凝抑制试验、IgM和IgG抗体捕获ELISA法等检测时存在交叉反应,结果易出现假阳性,并且,血清学检测方法也不能用于早期诊断。病毒分离是登革病毒感染诊断的金标准,并可进一步用于病毒血清型的鉴定,但是该方法费时而且对于实验室的条件要求较高。RT-PCR敏感性高,并且可以鉴定病毒血清型,但因技术要求高、样品易受污染且价格昂贵而使其应用受到限制。因此,需要建立一种简便快速的检测方法用于登革病毒感染的早期分型诊断。迄今为止,仅有本室利用DV1NS1特异性单抗成功建立双抗体夹心ELISA法用于检测病人血清中的DV1NS1抗原,实现了对DV1感染的早期快速分型诊断。登革病毒是一种单股正链RNA病毒,长度11kb,编码3种结构蛋白(C,prM/M,E)和7种非结构蛋白(NS1,NS2A,NS2B,NS3,NS4A,NS4B,NS5),结构蛋白组成的病毒外膜呈二十面立体对称体,非结构蛋白作为病毒复制复合物的一部分,主要是参与病毒RNA的复制。其中,NS1是DV一种重要的高度保守的糖蛋白,含有一个包含12个连续的半胱氨酸残基的恒定区,具有群特异性和型特异性决定簇,有胞内型、膜型和分泌型,其抗原性很强,含有多个T、B细胞表位,能够诱发细胞和体液免疫应答,且研究发现与DHF/DSS的发病机制有关。有研究发现在登革热患者的早期血中存在高浓度的NS1循环抗原,而检测NS1-IgG可初步进行登革病毒的分型,而本室已获得一组DV1NS1特异性单抗,并成功建立了以单克隆抗体为基础的双抗体夹心捕获DV1NS1抗原的酶联免疫学方法。鉴于登革热流行区大多同时存在4型登革病毒流行,发生不同血清型重复感染的危险性不断增加的特点,为研究DV3感染的早期诊断方法,本研究通过对DV3NS1基因的克隆、蛋白表达及其抗原性鉴定,并通过制备一组特异性抗Ⅲ型登革病毒NS1蛋白的单克隆抗体,建立以单克隆抗体为基础的双抗体夹心捕获DV3NS1抗原的酶联免疫学方法。本研究主要分为三个部分: 第一部分:Ⅲ型登革病毒NS1基因克隆及抗原性鉴定用RT-PCR方法扩增Ⅲ型登革病毒NS1全长基因,并定向克隆至原核表达载体pQE31中,pQE31为携带6个组氨酸(His-6)标签的融合蛋白表达载体,通过对表达条件和纯化条件的优化,经尿素和盐酸胍反复洗涤纯化成功获得融合蛋白,命名为DV3NS1。表达蛋白经Western Blot和ELISA鉴定,证明了可与登革病毒交叉性单抗结合,具有良好的抗原性,为进一步研究免疫诊断试剂奠定了基础。第二部分:Ⅲ型登革病毒NS1蛋白特异性单克隆抗体的制备及鉴定本部分研究在成功获得具有抗原性的DV3NS1蛋白的基础上,制备抗Ⅲ型登革病毒NS1蛋白特异性单抗,并对单抗进行免疫学特性研究、抗体识别抗原位点分析以及血清型特异性鉴定。采用重组DV3NS1蛋白与DV3全病毒混合交替免疫的方案分两批免疫BALB/c小鼠,取抗体效价高的小鼠脾脏与小鼠骨髓瘤细胞融合,对阳性的克隆细胞株进行有限稀释法亚克隆化,经分别以NS1蛋白和DV3病毒为抗原的两种间接ELISA筛选,结合免疫荧光鉴定,最终获得了26株稳定分泌抗NS1蛋白的单克隆抗体的杂交瘤细胞株。26株单抗Ig亚类测定,24株为IgG1,2株为IgG2a。ELISA和IFA鉴定6株特异结合Ⅲ型登革病毒NS1,与其余3型登革病毒不发生交叉反应,为血清型特异性单抗,而WesternBlot鉴定结果为阴性,初步说明它们识别的是构象型表位。经ELISA、WesternBlot和IFA鉴定有7株单抗与所有4型登革病毒交叉反应。采用相互竞争抑制试验分析单抗识别抗原表位,结果显示这一组单克隆抗体除了3株因生物素标记抗体效价很低而未行表位分析外,其余23株单抗可以识别7个以上不完全相同的抗原位点,并均能和天然病毒抗原结合,为下一步建立双抗体夹心抗原检测方法奠定了基础。第三部分:Ⅲ型登革病毒NS1抗原检测方法的建立根据本室Ⅰ型登革病毒抗原检测方法建立的途径,我们将26株单抗进行两两配对,以筛选出最佳抗体配对,用生物素标记每一株单抗,将每一株单抗作为捕获抗体分别与其它株生物素标记单抗作为检测抗体进行相互配对试验,通过检测登革病毒培养上清及检测NS1蛋白的灵敏度和特异性,结合检测正常人血的本底值高低,经反复多次筛选,最终选择了以血清型特异性单抗1D14A2A10为捕获抗体,型交叉性单抗5D32A17为检测抗体组成最佳的抗体对用于构建抗体夹心法。建立的双抗体夹心抗原捕获法检测重组NS1蛋白的灵敏度为0.4μg/ml。进一步检测不同血清型的登革病毒毒株和其它黄病毒的病毒培养液,结果显示,采用血清型特异性单抗作为捕获抗体,型交叉性单抗作为检测抗体建立的双抗体夹心抗原捕获法仅特异的检测到Ⅲ型登革病毒,而与其它病毒无交叉反应,检测方法具有型特异性。另外,由于目前缺乏Ⅲ型登革病毒感染病人血清,我们将模拟病人血清分为酸碱变性处理组和未处理组,用建立的双抗夹心ELISA检测模拟病人血清判断其检测血清中天然NS1蛋白的灵敏度,也发现未处理组的检测灵敏度明显较处理组要高,也进一步说明其中的捕获抗体识别的抗原表位是天然NS1蛋白的构像型表位。本部分研究表明,采用抗体夹心抗原捕获法建立的NS1蛋白检测方法具有较高的敏感性和特异性,将可应用于临床,实现对DV3感染的早期快速分型诊断。综合以上三个部分的研究结果,本研究的结论如下: 一、成功获得了具有良好抗原性的Ⅲ型登革病毒NS1重组蛋白,并进一步制备了26株具有血清型特异性和交叉性的Ⅲ型登革病毒NS1单克隆抗体,为免疫诊断试剂的研制、NS1蛋白功能的研究、登革病毒感染的发病机理及疫苗研制等奠定基础。二、本研究获得的26株单克隆抗体,经ELISA及IFA鉴定6株为DV3NS1特异性单抗,而Western Blot鉴定结果为阴性,初步推测其识别的抗原表位是构象型表位。而且,建立的Ⅲ型登革病毒夹心ELISA法检测变性重组NS1蛋白的灵敏度较天然DV3NS1蛋白低,与Western Blot检测结果一致,进一步说明检测方法中的捕获抗体识别的是天然NS1蛋白中的构像型表位,但还待于进一步证实。三、采用血清型特异性单抗作为捕获抗体和型交叉性单抗作为检测抗体,成功建立了Ⅲ型登革病毒双单克隆抗体夹心的NS1抗原检测方法,能特异的检测到Ⅲ型登革病毒中的NS1抗原,与其它3型登革病毒和相关病毒均无交叉反应,具有型特异性,有望应用于登革病毒感染的快速分型诊断或鉴别诊断。
[Abstract]:Dengue virus (DV) belongs to the yellow virus of the family yellicid family. Through the transmission of Aedes aegypti and Aedes albopictus, dengue virus includes 4 serotypes (DV1 to 4). Any type of dengue virus infection can cause a series of clinical symptoms, such as recessive infection, fever, dengue fever (DF), or more severe dengue hemorrhagic fever (DHF) and dengue shock heald. DSS. Dengue virus is mainly prevalent in tropical and subtropical regions. In nearly more than 100 countries, 2 billion 500 million people are threatened. In dengue epidemic areas, the number of type 4 dengue virus alternates, increasing the risk of repeated infection of different types. Dengue fever has become a mosquito borne disease, which is seriously harmful to human health. First infection is the first infection. The immunity obtained by the leather virus has a life-long immune protective effect on the reinfection of the same type of virus, but it can only produce partial and transient immune protection to the reinfection of other types of dengue virus. Because of the enhancement of antibody dependent infection, the reinfection of DV is the main risk of DHF/DSS. Risk factors. The present protective dengue virus vaccine has not been successfully developed, and there is no specific treatment for dengue virus infection in clinical. But the study shows that early clinical treatment can reduce the incidence and mortality of DHF. Since most dengue virus infected people lack specific clinical manifestations and DV infection is confirmed. Relying on laboratory diagnostic methods to isolate viruses from blood or tissue to detect virus antigens or RNA or to detect antibodies to serum specificity. Currently, the laboratory diagnostics of dengue virus mainly include virus isolation, serological detection and nucleic acid detection. Between 4 serotypes of dengue virus and other members of the yellow virus. There are common antigen epitopes. There are cross reactions in traditional serological methods such as hemagglutination inhibition test, IgM and IgG antibody capture ELISA. The results are false positive, and the serological detection method can not be used for early diagnosis. Virus isolation is the gold standard for the diagnosis of dengue virus infection and can be further used in the virus. The identification of serotypes, but this method is time-consuming, high.RT-PCR sensitivity to laboratory conditions, and can identify virus serotypes, but because of high technical requirements and high price, the application is limited. Therefore, a simple and rapid detection method is needed to be used for dengue virus infection. Early typing diagnosis. To date, only the double antibody sandwich ELISA method has been successfully established by DV1NS1 specific monoclonal antibody in the laboratory to detect DV1NS1 antigen in the patient's serum, and the early rapid typing diagnosis of DV1 infection is achieved.
Dengue virus (dengue virus) is a single strand positive chain RNA virus, length 11KB, encoding 3 structural proteins (C, prM/M, E) and 7 non structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The outer membrane of the structural protein is twenty faceted stereosymmetric body, and the non structural protein is part of the replication complex of the virus, which is mainly involved in the replication of the virus. NS1 is an important highly conserved glycoprotein of DV, containing a constant region containing 12 continuous cysteine residues, with group specific and type specific determinants, intracellular, membranous and secretory forms, with strong antigenicity, and multiple T, B cell epitopes, which can induce cellular and humoral immune responses, and research and DHF/DSS A high concentration of NS1 circulating antigen was found in the early blood of dengue patients, and the detection of NS1-IgG can preliminarily form the genotyping of dengue virus, and a group of DV1NS1 specific monoclonal antibodies have been obtained in this room, and the enzyme linked immunosorbent assay based on the monoclonal antibody sandwich DV1NS1 antigen is successfully established. In view of the prevalence of dengue virus type 4 dengue virus in the dengue epidemic area and the increasing risk of different serotype reinfections, in order to study the early diagnostic methods of DV3 infection, this study was conducted by cloning, protein expression and antigen identification of DV3NS1 gene, and preparing a group of specific anti - III types. A monoclonal antibody based monoclonal antibody based on double antibody sandwich for capturing DV3NS1 antigen was established by enzyme-linked immunosorbent assay (NS1).
This study is divided into three parts:
Part I: cloning and antigenicity identification of NS1 gene of dengue virus type III
The full length gene of type III dengue virus NS1 was amplified by RT-PCR and directed to the prokaryotic expression vector pQE31. PQE31 was a fusion protein carrier carrying 6 histidine (His-6) tags. The fusion protein was successfully obtained by repeated purification and purification of urea and guanidine hydrochloride by the optimization of the expression conditions and purification conditions. The fusion protein was named as DV3NS1. table. The protein was identified by Western Blot and ELISA, which proved that it could be combined with dengue virus cross monoclonal antibody, and had good antigenicity. It laid the foundation for further study of immunodiagnostic reagents.
The second part: preparation and identification of type III monoclonal antibodies against NS1 protein of dengue virus.
In this part, on the basis of the successful acquisition of antigenicity DV3NS1 protein, the specific monoclonal antibody against NS1 protein of type III dengue virus was prepared, and the immunological characteristics of the monoclonal antibody, the analysis of antibody identification antigen site and the specificity of serotype were identified. The alternative immunization scheme of recombinant DV3NS1 egg white and DV3 whole virus was divided into two Immunized BALB/c mice, the spleen of mice with high antibody titer was fused with murine myeloma cells, and the positive cloned cell lines were cloned by finite dilution method. Two indirect ELISA samples were screened with NS1 protein and DV3 virus as antigen respectively. In combination with immunofluorescence identification, 26 stable secreting anti NS1 proteins were obtained. Antibody hybridoma cell line.26 monoclonal antibody Ig subclass determination, 24 strains of IgG1,2 strains for IgG2a.ELISA and IFA identification of 6 specific binding type dengue virus NS1, and the remaining 3 type dengue virus no cross reaction, the serotype specific monoclonal antibody, and WesternBlot identification results are negative, preliminarily indicated that they identified conformational epitopes. Via ELIS A, WesternBlot and IFA identified the cross reaction between 7 McAbs and all type 4 dengue viruses. The monoclonal antibody epitopes were analyzed by competitive inhibition test. The results showed that the group of monoclonal antibodies could identify more than 7 incompletely identical monoclonal antibodies in addition to the low epitope analysis of 3 biotin labeled antibodies. The antigen sites can be combined with the natural virus antigen, which lays the foundation for the establishment of double antibody sandwich antigen detection method in the next step.
The third part: establishment of detection method for type III dengue virus NS1 antigen.
According to the way of establishing the antigen detection method of dengue virus type I in this room, we paired 26 McAbs with 22 pairs to screen out the best antibody pairs. Each monoclonal antibody was labeled with biotin, and each mAb was paired as a capture antibody and other biotin labeled monoclonal antibody as a test antibody. With the sensitivity and specificity of dengue virus culture and detection of NS1 protein, combined with the detection of the background value of normal human blood, after repeated screening, the final selection of the serotype specific monoclonal antibody 1D14A2A10 as the capture antibody and the cross monoclonal antibody 5D32A17 as the best antibody for the detection of antibodies is used to construct the antibody sandwich method. The sensitivity of the double antibody sandwich antigen capture method to detect the recombinant NS1 protein was 0.4 micron g/ml. for further detection of different serotype dengue virus strains and other virus cultures of other yellow viruses. The results showed that the serotype specific monoclonal antibody was used as the capture antibody and the type of cross monoclonal antibody was used as a double antibody sandwich antigen to detect antibody. The method was only specific to detect type III dengue virus, but no cross reaction with other viruses, the detection method was specific. In addition, due to the lack of sera of patients with type III dengue virus infection, we divided the simulated patients' serum into acid base denaturation treatment group and untreated group, and used the established double anti sandwich ELISA to detect the serum of simulated patients. The sensitivity of the natural NS1 protein in the serum was detected, and the detection sensitivity of the untreated group was significantly higher than that in the treatment group. It also indicated that the antigen epitopes identified by the captured antibody were the epitopes of the natural NS1 protein. This part of the study showed that the detection method of NS1 protein based on the antibody sandwich antigen capture method was used. It has high sensitivity and specificity, and will be applied in clinic to achieve rapid typing diagnosis of DV3 infection.
Based on the above three parts, the conclusions of this study are as follows:
First, the recombinant protein of type III dengue virus NS1 with good antigenicity was successfully obtained, and 26 monoclonal antibodies with serotype specific and cross type of dengue virus NS1 were prepared, which lay the foundation for the development of immuno diagnostic reagents, the study of the function of NS1 protein, the pathogenesis of dengue virus infection and the development of the vaccine.
Two, 26 monoclonal antibodies obtained by this study were identified by ELISA and IFA as DV3NS1 specific monoclonal antibodies, and the results of Western Blot identification were negative. It was preliminarily conjectured that the identified epitopes were conformation epitopes. Moreover, the sensitivity of the established type III dengue virus sandwich ELISA method to detect denatured recombinant NS1 protein was lower than that of natural DV3NS1 protein. The results of Western Blot detection are consistent, which further illustrates that the capture antibody identification in the detection method is the structure epitope of the natural NS1 protein, but it remains to be further confirmed.
Three, using the serotype specific monoclonal antibody as the capture antibody and the type cross monoclonal antibody as the detection antibody, the NS1 antigen detection method of the type III dengue virus double monoclonal antibody sandwich was successfully established. It could detect the NS1 antigen in the type III dengue virus specifically, and had no cross reaction with other type 3 dengue virus and related viruses. Specificity is expected to be applied to rapid typing diagnosis or differential diagnosis of dengue virus infection.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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