马尔堡病毒抗原、抗体ELISA检测方法的建立与评价

发布时间：2018-04-20 11:24

本文选题：马尔堡出血热 + 马尔堡病毒　；参考：《吉林农业大学》2017年硕士论文

【摘要】：马尔堡出血热(Marburg Hemorrhagic Fever,MHF)也称为马尔堡病毒病,是由马尔堡病毒(Marburg virus,MARV)引起的严重出血热的人兽共患传染病,发病后几乎高达90%的死亡率,至今仍无商品化的治疗药物和疫苗。该病毒首次在德国的马尔堡城市的实验室内分离并因此被命名。大多数严重的病例主要发生在医疗设备相对落后的非洲。马尔堡出血热主要通过密切接触传播,因此应该对病人及可疑病例进行隔离,建立相关的快速安全的检测方法是控制该病传播的最基本的手段。MARV的基因组由单股负链RNA构成,病毒基因组全长约19.1kb,共包括7个阅读框(ORF),分别编码7种蛋白:核蛋白(NP)、包膜糖蛋白(GP)、结构蛋白(VP30和VP35)、基质蛋白VP24和VP40、RNA依赖的RNA多聚酶(L蛋白)。该病毒目前只发现一个血清型。其中,MARV糖蛋白(Glycoprotein,GP)是病毒仅有的表面蛋白,能够诱导机体产生中和抗体。GP由GP1和GP2构成,受体结合域(RBD,aa38-188)是位于GP1内N端的一个不连续的独立折叠区域,能够比完整的GP1更好的与细胞受体结合产生反应。本研究利用大肠杆菌原核表达系统表达重组MARV-GP-RBD蛋白,并以该蛋白为基础建立MARV抗体间接ELISA方法并制备抗MARV-GP-RBD蛋白的单克隆抗体及多克隆抗体;建立了马尔堡病毒样颗粒糖蛋白含量检测双抗体夹心ELISA方法。原核表达马尔堡病毒糖蛋白受体结合域及纯化并制备单克隆抗体及多克隆抗体。目的原核表达并纯化马尔堡病毒(Maburg Virus,MARV)糖蛋白(Glycoprotein,GP)的受体结合域(Receptor Binding Domain,RBD)蛋白,以之为抗原免疫家兔制备多克隆抗体,免疫小鼠制备单克隆抗体。方法参照GenBank提供的MARV GP全基因序列,找到主要抗原表位区域(Receptor Binding Domain,RBD),设计特异性引物,采用PCR方法扩增RBD基因,扩增产物经双酶切(EcoRⅠ/XhoⅠ)后定向克隆至原核表达载体pET-30a(+),构建重组表达质粒pET-30a(+)-GP-RBD,转化BL21(DE3)感受态表达宿主菌,在不同条件下(时间、IPTG浓度、温度)诱导表达目的蛋白,并用His-Band N+柱进行亲和层析纯化;以纯化的重组pET-30a(+)-GP-RBD蛋白免疫家兔和小鼠,制备多克隆抗体和单克隆抗体。通过SDS-PAGE、Western blot和IFA鉴定目的蛋白的反应原性和免疫原性。结果PCR扩增到长度为453bp的RBD基因片段;构建的重组质粒pET-30a(+)-GP-RBD经双酶切后得到与目的片段长度相同的特异性条带,测序结果显示没有突变;转化产物在培养7h、终浓度为0.4mmol/l IPTG和37℃条件下能够充分诱导目的蛋白表达,得到分子质量大小为25ku的重组蛋白,主要以包涵体形式存在,BCA试剂盒产量测定,每升诱导的重组菌可纯化约20mg左右纯度较高的目的蛋白;Western blot检测证实了重组pET-30a(+)-GP-RBD蛋白能同时被抗His标签的单抗、兔源多抗和鼠源单抗识别并发生特异性反应,证明该蛋白具有较好的反应原性;IFA鉴定证实所制备的兔源多抗和鼠源单抗均能够特异性识别表达MARV GP蛋白的重组杆状病毒rBacmid-GP-VP40,证明重组蛋白具有良好的免疫原性。结论成功表达纯化了MARV GP蛋白RBD蛋白,并完成了兔源多抗和鼠源单抗的制备与鉴定,为MARV亚单位疫苗的制备和抗原、抗体检测方法的建立奠定了基础。间接ELISA检测马尔堡病毒抗体方法的建立及初步应用。目的建立MARV抗体间接ELISA检测方法,并对所建立方法的相关条件进行了优化。方法以纯化后的MARV糖蛋白的受体结合域蛋白作为包被抗原,MARV病毒样颗粒免疫马匹获得的高免马血清为一抗,MARV阴性马血清为阴性对照,HRP标记山羊抗马IgG为二抗,优化反应条件并评价其特异性和敏感性。结果抗原的最佳包被质量浓度为4ug/ml;血清最佳稀释度为1:640;酶标二抗的最佳浓度为1:5000;优化后的间接ELISA方法可特异性检测MARV抗体,与埃博拉病毒,西尼罗河热病毒,委内瑞拉马脑炎,裂谷热等病毒的阳性血清没有交叉反应。批内、批间试验变异系数均小于10%。结论本研究成功建立了MARV抗体检测间接ELISA方法,为MARV的血清学调查奠定基础。双抗体夹心ELISA定量检测马尔堡病毒糖蛋白检测方法的建立及初步应用。目的定量检测马尔堡病毒病毒样颗粒糖蛋白含量。方法以纯化的MARV GP A21单克隆抗体为捕获抗体,纯化的A1单抗为检测抗体,纯化的原核表达的MARV GP作为标准品绘制标准曲线。优化并选择最佳作用条件并对建立的方法进行评价。结果A21抗体最佳稀释度为2560(包被浓度为0.742ug/ml),HRP标记后的A1最佳稀释度为1:8000。与埃博拉病毒,西尼罗河热病毒,裂谷热病毒,中东呼吸综合征病毒等无交叉反应,批内、批间试验变异系数均小于10%,对MARV的检测灵敏度达21.052 ng/ml。结论该方法可特异性定量检测MARV,与其他病毒均不发生反应。该方法灵敏度、特异性高、重复性好。该方法可对马尔堡病毒样颗粒中的糖蛋白的相对含量进行监测。该方法适用于马尔堡病毒样颗粒疫苗免疫剂量的测定。
[Abstract]:Marburg Hemorrhagic Fever (MHF), also known as Marburg virus disease, is a human zoonosis of severe hemorrhagic fever caused by the Marburg virus (MARV), almost 90% of mortality after the onset of the disease, and there is still no commercialized treatment and vaccine. The virus was first in the Marburg city of Germany. Most serious cases mainly occur in relatively backward Africa. The Marburg haemorrhagic fever is mainly transmitted through close contact and therefore should be isolated from patients and suspected cases, and the establishment of a related rapid and safe method of detection is the most basic means of controlling the transmission of the disease,.MARV. The genome is composed of single strand negative chain RNA. The total length of the virus genome is about 19.1kb, including 7 reading frames (ORF), which encode 7 proteins: nucleoprotein (NP), membrane glycoprotein (GP), structural protein (VP30 and VP35), matrix protein VP24 and VP40, RNA dependent RNA polyenzyme (L protein). The virus currently only found one serotype. Oprotein, GP) is the only surface protein of the virus, which can induce the organism to produce neutralizing antibody.GP, which is composed of GP1 and GP2. The receptor binding domain (RBD, aa38-188) is an independent, discontinuous folding region located at the N end of GP1, which can better react with the cell receptor than the complete GP1. This study uses the Escherichia coli prokaryotic expression system. The recombinant MARV-GP-RBD protein was expressed and the indirect ELISA method of MARV antibody was established on the basis of the protein, and the monoclonal antibody and polyclonal antibody against MARV-GP-RBD protein were prepared. A double antibody sandwich ELISA method was established for the detection of Marburg virus like granular glycoprotein content. The binding domain of the glycoprotein receptor of Marburg virus was expressed and purified. Preparation of monoclonal antibodies and polyclonal antibodies. Aim prokaryotic expression and purification of the receptor binding domain (Receptor Binding Domain, RBD) of Maburg Virus (MARV) glycoprotein (Glycoprotein, GP). The polyclonal antibody was prepared for antigen immunization in rabbits, and monoclonal antibodies were prepared from the immunized mice. The method referred to MARV GP provided by GenBank. The whole gene sequence, Receptor Binding Domain (RBD) was found, specific primers were designed, RBD gene was amplified by PCR, and the amplified products were cloned to the prokaryotic expression vector pET-30a (+) after double enzyme digestion (EcoR I /Xho I), and the recombinant plasmid was constructed to plasmid pET-30a (+) -GP-RBD, and the host bacteria were transformed into the receptive state of the BL21. The target protein was induced under different conditions (time, IPTG concentration, temperature) and purified by affinity chromatography with His-Band N+ column, and the purified recombinant pET-30a (+) -GP-RBD protein was used to immunize rabbits and mice to prepare polyclonal and monoclonal antibodies. The reactivity and immunogenicity of the target protein were identified by SDS-PAGE, Western blot and IFA. Results PCR amplified the RBD gene fragment length of 453bp, and the recombinant plasmid pET-30a (+) -GP-RBD obtained the same specific bands as the length of the target fragment after double enzyme digestion. The sequencing results showed no mutation, and the transformed product could fully induce the expression of the target protein in the culture of 7h, the final concentration of 0.4mmol/l IPTG and 37 C. The recombinant protein with a molecular weight of 25ku, mainly in inclusion body form, determination of BCA kit yield, and purified recombinant bacteria with high purity about 20mg about 20mg per liter, and Western blot detection confirmed that the recombinant pET-30a (+) -GP-RBD protein can be simultaneously identified with anti His labelling monoclonal antibody, rabbit source polyclonal and mouse monoclonal antibody recognition and The specific reaction showed that the protein had good reactivity. The IFA identification confirmed that the rabbit source polyclonal and mouse McAbs were able to identify the recombinant baculovirus rBacmid-GP-VP40 specifically expressing MARV GP protein, which proved that the recombinant protein had good immunogenicity. The MARV GP protein RBD protein was successfully expressed and purified. The preparation and identification of rabbit source polyclonal and mouse monoclonal antibody were completed. The basis was laid for the preparation and antigen of MARV subunit vaccine and the establishment of antibody detection methods. The establishment and preliminary application of the indirect ELISA method for detecting the antibody of Marburg virus was established. Objective to establish the indirect ELISA detection method for MARV antibody, and to carry out the related conditions of the established method. Methods using the purified MARV glycoprotein receptor binding domain protein as the inclusion antigen, the high free horse serum obtained by the MARV virus like particles, the MARV negative horse serum as the negative control, the HRP labeled Goat anti horse IgG as two, the optimum reaction conditions and the evaluation of its specificity and sensitivity. The mass concentration was 4ug/ml, the best dilution of serum was 1:640, the best concentration of anti enzyme two was 1:5000, and the optimized indirect ELISA method was specific to detect MARV antibody. There was no cross reaction with Ebola virus, West Nile heat virus, Venezuela horse encephalitis, rift heat and so on. The coefficient of variation in batch test was small. In 10%. conclusion, the indirect ELISA method for the detection of MARV antibody was established successfully, which laid the foundation for the serological investigation of MARV. The quantitative detection of Marburg virus like glycoprotein content by double antibody sandwich ELISA quantitative detection of Marburg virus glycoprotein. Method for the purification of the purified MARV GP A21 monomer. The cloned antibody was captured. The purified A1 monoclonal antibody was the detection antibody and the purified prokaryotic expression MARV GP was used as standard curve. The optimum conditions were optimized and selected to evaluate the established method. The best dilution degree of A21 antibody was 2560 (the concentration was 0.742ug/ml), and the best dilution degree of A1 after HRP was 1:8000. There was no cross reaction with Ebola virus, West Nile heat virus, Rift Valley fever virus, Middle East respiratory syndrome virus and so on. In batch, the coefficient of variation was less than 10% in batch test, and the sensitivity of MARV was 21.052 ng/ml.. Conclusion the method can detect MARV with specificity and no reaction to his virus. The sensitivity and specificity of this method is high. The method can be used to monitor the relative content of glycoproteins in Marburg virus like particles. This method is suitable for the determination of the immune dose of Marburg virus like particle vaccine.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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