EV71型肠道病毒抗体的制备与鉴定

发布时间：2019-01-01 20:18

【摘要】：肠道病毒71型(Enterovirus type71, EV71)是小型RNA病毒科肠道病毒属成员,其感染性强、致病率高,是导致手足口病的主要病原,同时能引起多种与神经系统相关的疾病。由于肠道病毒属成员数量多、结构和生物学特性相似,导致其临床诊断和分类鉴定难度加大。目前针对肠道病毒71型感染多采用抗生素进行治疗,但是在当前病毒耐药性不断增加的情况下,寻找一种更安全、更高效的治疗特效药及诊断方法成为一个急需解决的问题。本论文实验：(1)制备特异性抗肠道病毒71型卵黄抗体,然后通过SDS-PAGE凝胶电泳、考马斯亮蓝染色法、酶联免疫吸附试验、免疫双向琼脂扩散、Western blot、体外病毒中和实验等方法对其生物活性进行鉴定;(2)通过建立阳性杂交瘤细胞株及体内诱生腹水法制备了大量抗肠道病毒71型VP1蛋白单克隆抗体,辛酸硫酸铵沉淀法纯化后通过SDS-PAGE电泳和紫外分光光度法鉴定抗体纯度和含量,间接ELISA法测定其中和效价,鼠单抗业型分型试纸鉴定其Ig类与业类;(3)利用偶联剂EDC和Sulfo-NHS将肠道病毒71型VP1蛋白单克隆抗体偶联到聚丙烯酸修饰的水溶性核量子点上,并利用酶联免疫法、荧光发射光谱、高性能生物质谱等分析方法对偶联物进行分析。在此基础上应用卵黄抗体为捕获抗体,量子点标记的单克隆抗体作为检测抗体,建立了量子点标记免疫荧光检测EV71病毒的标准曲线。实验结果：(1)考马斯亮蓝染色法测得卵黄中IgY得率达7.76mg/mL,SDS-PAGE凝胶电泳测得IgY抗体的纯度为94.86%。初免10d后蛋黄中可检测到特异性抗EV71IgY抗体,初免40天后ELISA检测抗体效价达最高1：20480,双向琼脂扩散检测效价达1:16。Western blot分析提取的IgY抗体具有良好的免疫反应性,能与EV71特异性结合；同时制备的特异性抗EV71IgY具有良好的体外中和活性,最低中和溶度为7.9ug/mL,中和效价为1：128。(2)初步筛选到2株抗肠道病毒71型VPl蛋白的细胞株,效价均达到了106以上；腹水纯化后经还原性SDS-PAGE电泳可见两条清晰的IgG重轻链,紫外分光光度法测纯化前后蛋白浓度分别为为39.28mg/mL和5.6mg/mL。经鼠单抗业型分型试纸鉴定获得的单抗类型为IgG2b类,轻链为K链。(3)由酶联免疫法及紫外透射反射观察结果可知QDS-MAb同时拥有MAb的生物活性及QDS的化学发光特性。荧光光谱图中QDS-MAb荧光最大发射波长红移4nm及质谱图中QDS-MAb的分子离子峰比MAb分子离子峰增大了2973这些结果都证实抗肠道病毒71型VP1单克隆抗体蛋白成功偶联到水溶性量子点上,且结构未受破坏。通过棋盘滴定法确定了量子点荧光免疫检测EV71法中卵黄抗体最佳包被溶度为4ng/mL,病毒检测标准曲线方程为y=301.66x+3307,R2=0.9911,检测范围为1.953×10-3ug/mL至0.03125ug/mL
[Abstract]:Enterovirus 71 (Enterovirus type71, EV71) is a member of the genus of enterovirus in the family RNA, which is highly infectious and highly pathogenic. It is the main cause of hand, foot and mouth disease and can cause many diseases related to the nervous system at the same time. Because of the large number of enterovirus members and the similar structure and biological characteristics, the clinical diagnosis and classification of enterovirus become more difficult. At present, antibiotics are often used to treat enterovirus 71 infection. However, with the increasing drug resistance, it is urgent to find a more safe and efficient method for the treatment and diagnosis of enterovirus 71 infection. In this paper: (1) preparation of specific anti-enterovirus type 71 egg yolk antibody, then through SDS-PAGE gel electrophoresis, Coomassie brilliant blue staining, enzyme-linked immunosorbent assay, immunized two-way Agar diffusion, Western blot, Its biological activity was identified by virus neutralization test in vitro. (2) A large number of monoclonal antibodies against enterovirus 71 VP1 protein were prepared by establishing a positive hybridoma cell line and inducing ascites in vivo. The purity and content of antibody were identified by SDS-PAGE electrophoresis and UV spectrophotometry after purification by ammonium octanoate precipitation method. The titer of the antibody was determined by indirect ELISA method. The Ig class and industry class were identified by mouse monoclonal antibody typing test paper. (3) the monoclonal antibody against enterovirus 71 VP1 protein was conjugated to the water-soluble nuclear quantum dot modified by polyacrylic acid by coupling agents EDC and Sulfo-NHS, and the fluorescence emission spectrum was determined by enzyme-linked immunosorbent assay (Elisa). The coupling compounds were analyzed by high performance biological mass spectrometry. On this basis, using yolk antibody as capture antibody and quantum dot labeled monoclonal antibody as detection antibody, the standard curve of EV71 virus detection using quantum dot labeled immunofluorescence was established. The results were as follows: (1) the yield of IgY in yolk by Coomassie brilliant blue staining was 7.76 mg / mL SDS-PAGE and the purity of IgY antibody was 94.86. Specific anti EV71IgY antibody could be detected in egg yolk 10 days after the first immunization, and the titer of ELISA antibody reached 1: 20480 after 40 days, and the titer of IgY antibody extracted by 1:16.Western blot analysis by bidirectional Agar diffusion assay had good immunoreactivity. Can specifically bind to EV71; At the same time, the specific anti- EV71IgY prepared had good neutralization activity in vitro, the lowest neutralization solubility was 7.9 ugmL, and the neutralization titer was 1: 128. (2) two cell lines against enterovirus 71 VPl protein were preliminarily screened. The titers were above 106; After purification of ascites, two clear IgG heavy and light chains were found by reductive SDS-PAGE electrophoresis. The protein concentrations were 39.28mg/mL and 5.6 mg / mL before and after purification by UV spectrophotometry. The type of monoclonal antibody was identified as IgG2b and light chain was K chain. (3) the results of enzyme-linked immunosorbent assay and ultraviolet transmission reflectance showed that QDS-MAb possessed the bioactivity of MAb and the chemiluminescence characteristics of QDS at the same time. The maximum emission wavelength of QDS-MAb red shift 4nm in fluorescence spectrum and the molecular ion peak of QDS-MAb in mass spectrogram are 2973 larger than that of MAb. These results confirm that the monoclonal antibody protein against enterovirus 71 VP1 has been successfully coupled. Connected to water-soluble quantum dots, And the structure is not damaged. By means of chessboard titration, the best coating solubility of egg yolk antibody in EV71 was determined to be 4ng / mL, the standard curve equation of virus detection was yang301.66x 3307R2O0.9911, and the detection range was from 1.953 脳 10-3ug/mL to 0.03125ug/mL.
【学位授予单位】：广东工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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