發(fā)布時間：2018-06-08 10:49

  本文選題：III型登革病毒 + 非結(jié)構(gòu)蛋白1��； 參考：《南方醫(yī)科大學》2008年碩士論文

【摘要】： 登革病毒(DV)屬于黃病毒科黃病毒屬,通過埃及伊蚊和白蚊伊蚊傳播,登革病毒包括4種血清型(DV1～4),任何型別的登革病毒感染均可引起一系列的臨床癥狀,表現(xiàn)為隱性感染、發(fā)熱、登革熱(DF)、或更為嚴重的登革出血熱(DHF)和登革休克綜合征(DSS)。登革病毒主要流行于熱帶和亞熱帶地區(qū),近100多個國家、25億人口受到威脅,在登革疫區(qū)中,多見4型登革病毒交替流行,更增加了不同型別反復感染的危險性。登革熱已成為一種嚴重危害人類健康的蚊媒病毒傳染病。初次感染登革病毒所獲得的免疫力對于同型病毒的再次感染可產(chǎn)生終身的免疫保護作用,但是對于其它型別登革病毒的再次感染僅能產(chǎn)生部分而且短暫的免疫保護作用。由于存在抗體依賴的感染增強作用,異型DV再次感染是發(fā)生DHF/DSS的主要危險因素。目前具有保護性的登革病毒疫苗尚未研制成功,臨床上對于登革病毒的感染亦未有特異性的治療措施。但研究表明及早的臨床處理可以減少DHF的發(fā)病率和致死率。由于多數(shù)登革病毒感染者早期缺乏特異的臨床表現(xiàn),DV感染確診依賴于實驗室診斷手段,從血液或組織中分離到病毒,檢測病毒抗原或RNA或者檢測血清特異性的抗體。目前,登革病毒的實驗室診斷方法主要包括病毒分離、血清學檢測和核酸檢測。由于4種登革病毒的血清型之間,以及與其它黃病毒成員間存在共同抗原表位,用傳統(tǒng)的血清學方法如血凝抑制試驗、IgM和IgG抗體捕獲ELISA法等檢測時存在交叉反應(yīng),結(jié)果易出現(xiàn)假陽性,并且,血清學檢測方法也不能用于早期診斷。病毒分離是登革病毒感染診斷的金標準,并可進一步用于病毒血清型的鑒定,但是該方法費時而且對于實驗室的條件要求較高。RT-PCR敏感性高,并且可以鑒定病毒血清型,但因技術(shù)要求高、樣品易受污染且價格昂貴而使其應(yīng)用受到限制。因此,需要建立一種簡便快速的檢測方法用于登革病毒感染的早期分型診斷。迄今為止,僅有本室利用DV1NS1特異性單抗成功建立雙抗體夾心ELISA法用于檢測病人血清中的DV1NS1抗原,實現(xiàn)了對DV1感染的早期快速分型診斷。 登革病毒是一種單股正鏈RNA病毒,長度11kb,編碼3種結(jié)構(gòu)蛋白(C,prM/M,E)和7種非結(jié)構(gòu)蛋白(NS1,NS2A,NS2B,NS3,NS4A,NS4B,NS5),結(jié)構(gòu)蛋白組成的病毒外膜呈二十面立體對稱體,非結(jié)構(gòu)蛋白作為病毒復制復合物的一部分,主要是參與病毒RNA的復制。其中,NS1是DV一種重要的高度保守的糖蛋白,含有一個包含12個連續(xù)的半胱氨酸殘基的恒定區(qū),具有群特異性和型特異性決定簇,有胞內(nèi)型、膜型和分泌型,其抗原性很強,含有多個T、B細胞表位,能夠誘發(fā)細胞和體液免疫應(yīng)答,且研究發(fā)現(xiàn)與DHF/DSS的發(fā)病機制有關(guān)。有研究發(fā)現(xiàn)在登革熱患者的早期血中存在高濃度的NS1循環(huán)抗原,而檢測NS1-IgG可初步進行登革病毒的分型,而本室已獲得一組DV1NS1特異性單抗,并成功建立了以單克隆抗體為基礎(chǔ)的雙抗體夾心捕獲DV1NS1抗原的酶聯(lián)免疫學方法。鑒于登革熱流行區(qū)大多同時存在4型登革病毒流行,發(fā)生不同血清型重復感染的危險性不斷增加的特點,為研究DV3感染的早期診斷方法,本研究通過對DV3NS1基因的克隆、蛋白表達及其抗原性鑒定,并通過制備一組特異性抗Ⅲ型登革病毒NS1蛋白的單克隆抗體,建立以單克隆抗體為基礎(chǔ)的雙抗體夾心捕獲DV3NS1抗原的酶聯(lián)免疫學方法。 本研究主要分為三個部分: 第一部分:Ⅲ型登革病毒NS1基因克隆及抗原性鑒定 用RT-PCR方法擴增Ⅲ型登革病毒NS1全長基因,并定向克隆至原核表達載體pQE31中,pQE31為攜帶6個組氨酸(His-6)標簽的融合蛋白表達載體,通過對表達條件和純化條件的優(yōu)化,經(jīng)尿素和鹽酸胍反復洗滌純化成功獲得融合蛋白,命名為DV3NS1。表達蛋白經(jīng)Western Blot和ELISA鑒定,證明了可與登革病毒交叉性單抗結(jié)合,具有良好的抗原性,為進一步研究免疫診斷試劑奠定了基礎(chǔ)。 第二部分:Ⅲ型登革病毒NS1蛋白特異性單克隆抗體的制備及鑒定 本部分研究在成功獲得具有抗原性的DV3NS1蛋白的基礎(chǔ)上,制備抗Ⅲ型登革病毒NS1蛋白特異性單抗,并對單抗進行免疫學特性研究、抗體識別抗原位點分析以及血清型特異性鑒定。采用重組DV3NS1蛋白與DV3全病毒混合交替免疫的方案分兩批免疫BALB/c小鼠,取抗體效價高的小鼠脾臟與小鼠骨髓瘤細胞融合,對陽性的克隆細胞株進行有限稀釋法亞克隆化,經(jīng)分別以NS1蛋白和DV3病毒為抗原的兩種間接ELISA篩選,結(jié)合免疫熒光鑒定,最終獲得了26株穩(wěn)定分泌抗NS1蛋白的單克隆抗體的雜交瘤細胞株。26株單抗Ig亞類測定,24株為IgG1,2株為IgG2a。ELISA和IFA鑒定6株特異結(jié)合Ⅲ型登革病毒NS1,與其余3型登革病毒不發(fā)生交叉反應(yīng),為血清型特異性單抗,而WesternBlot鑒定結(jié)果為陰性,初步說明它們識別的是構(gòu)象型表位。經(jīng)ELISA、WesternBlot和IFA鑒定有7株單抗與所有4型登革病毒交叉反應(yīng)。采用相互競爭抑制試驗分析單抗識別抗原表位,結(jié)果顯示這一組單克隆抗體除了3株因生物素標記抗體效價很低而未行表位分析外,其余23株單抗可以識別7個以上不完全相同的抗原位點,并均能和天然病毒抗原結(jié)合,為下一步建立雙抗體夾心抗原檢測方法奠定了基礎(chǔ)。 第三部分:Ⅲ型登革病毒NS1抗原檢測方法的建立 根據(jù)本室Ⅰ型登革病毒抗原檢測方法建立的途徑,我們將26株單抗進行兩兩配對,以篩選出最佳抗體配對,用生物素標記每一株單抗,將每一株單抗作為捕獲抗體分別與其它株生物素標記單抗作為檢測抗體進行相互配對試驗,通過檢測登革病毒培養(yǎng)上清及檢測NS1蛋白的靈敏度和特異性,結(jié)合檢測正常人血的本底值高低,經(jīng)反復多次篩選,最終選擇了以血清型特異性單抗1D14A2A10為捕獲抗體,型交叉性單抗5D32A17為檢測抗體組成最佳的抗體對用于構(gòu)建抗體夾心法。建立的雙抗體夾心抗原捕獲法檢測重組NS1蛋白的靈敏度為0.4μg/ml。進一步檢測不同血清型的登革病毒毒株和其它黃病毒的病毒培養(yǎng)液,結(jié)果顯示,采用血清型特異性單抗作為捕獲抗體,型交叉性單抗作為檢測抗體建立的雙抗體夾心抗原捕獲法僅特異的檢測到Ⅲ型登革病毒,而與其它病毒無交叉反應(yīng),檢測方法具有型特異性。另外,由于目前缺乏Ⅲ型登革病毒感染病人血清,我們將模擬病人血清分為酸堿變性處理組和未處理組,用建立的雙抗夾心ELISA檢測模擬病人血清判斷其檢測血清中天然NS1蛋白的靈敏度,也發(fā)現(xiàn)未處理組的檢測靈敏度明顯較處理組要高,也進一步說明其中的捕獲抗體識別的抗原表位是天然NS1蛋白的構(gòu)像型表位。本部分研究表明,采用抗體夾心抗原捕獲法建立的NS1蛋白檢測方法具有較高的敏感性和特異性,將可應(yīng)用于臨床,實現(xiàn)對DV3感染的早期快速分型診斷。 綜合以上三個部分的研究結(jié)果,本研究的結(jié)論如下: 一、成功獲得了具有良好抗原性的Ⅲ型登革病毒NS1重組蛋白,并進一步制備了26株具有血清型特異性和交叉性的Ⅲ型登革病毒NS1單克隆抗體,為免疫診斷試劑的研制、NS1蛋白功能的研究、登革病毒感染的發(fā)病機理及疫苗研制等奠定基礎(chǔ)。 二、本研究獲得的26株單克隆抗體,經(jīng)ELISA及IFA鑒定6株為DV3NS1特異性單抗,而Western Blot鑒定結(jié)果為陰性,初步推測其識別的抗原表位是構(gòu)象型表位。而且,建立的Ⅲ型登革病毒夾心ELISA法檢測變性重組NS1蛋白的靈敏度較天然DV3NS1蛋白低,與Western Blot檢測結(jié)果一致,進一步說明檢測方法中的捕獲抗體識別的是天然NS1蛋白中的構(gòu)像型表位,但還待于進一步證實。 三、采用血清型特異性單抗作為捕獲抗體和型交叉性單抗作為檢測抗體,成功建立了Ⅲ型登革病毒雙單克隆抗體夾心的NS1抗原檢測方法,能特異的檢測到Ⅲ型登革病毒中的NS1抗原,與其它3型登革病毒和相關(guān)病毒均無交叉反應(yīng),具有型特異性,有望應(yīng)用于登革病毒感染的快速分型診斷或鑒別診斷。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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