流产衣原体MIP蛋白的双抗原夹心ELISA诊断方法建立及单克隆抗体制备

发布时间：2018-07-05 13:24

本文选题：巨噬细胞感染增强蛋白 + 双抗原夹心ELISA　；参考：《中国农业科学院》2015年硕士论文

【摘要】：流产衣原体(Chlamydia abortus,C.abortus)是一种严格细胞内寄生的革兰氏阴性细菌,可感染妊娠母畜导致流产、死产或者弱胎等;也可引起公畜发生睾丸炎、尿道炎等症状;孕妇与感染的动物直接接触也会造成流产及全身感染等症状,因此流产衣原体是一种重要的人兽共患病病原。巨噬细胞感染增强蛋白(MacropHage Infectivity Potentiator,MIP)是一种脂蛋白,作为一种免疫显性抗原,参与衣原体感染,引起炎症反应等过程。本研究以MIP为研究对象,利用Gen Bank已公布的序列,设计引物,PCR扩增得到MIP蛋白的编码基因,经过酶切、连接、转化将其插入原核表达载体pET30a(+),成功构建表达载体pET-mip并转化入E.coli BL21(DE3)中诱导表达,经SDS-PAGE分析可见在27 kD处出现特异性条带,表明插入的mip基因成功表达;Western-blot特异性实验验证表达的MIP蛋白具有反应原性。以纯化的MIP蛋白做为包被抗原,HRP标记的MIP蛋白为酶标抗原,最适包被浓度为2.5μg/mL,血清最佳稀释度为1:100,建立双抗原夹心ELISA方法。建立的双抗原ELISA与IHA相比,敏感性高,符合率为89.3%;与支原体、嗜肺军团菌、口蹄疫等阳性血清无交叉反应;批间、批内CV%均小于11%,表明建立的双抗原夹心ELISA方法特异性强、灵敏度高、重复性好。以纯化的MIP蛋白免疫Balb/c小鼠后筛选阳性细胞克隆并进行有限稀释克隆化,建立了两株能稳定分泌抗MIP蛋白单克隆抗体的杂交瘤细胞株,分别命名为M1和M3。间接ELISA法鉴定单抗上清抗体效价为1:1600;染色体鉴定符合杂交瘤细胞的特性;两株单抗50%最大结合浓度均为10μg/mL;Western-blot鉴定两株单克隆抗体能特异识别MIP蛋白;Mouse单克隆抗体亚类检测试剂盒鉴定2株单抗免疫球蛋白类型均为IgG1,轻链为κ链;鸡胚接种鉴定2株单抗能有效中和衣原体感染性。综上所述,本研究成功表达了流产衣原体MIP蛋白,建立了MIP蛋白双抗原夹心ELISA诊断方法,制备了抗MIP单克隆抗体,为流产衣原体的准确诊断及相关致病机制的研究和奠定了基础。
[Abstract]:Chlamydia abortus C. abortus is a strictly cell-parasitic gram-negative bacteria, which can infect pregnant females to cause abortion, stillbirth or weak fetus, and can also cause orchitis, urethritis and other symptoms in male animals. Direct contact between pregnant women and infected animals can also cause abortion and systemic infection, so chlamydia miscarriage is an important zoonotic pathogen. Macrophage infection Enhancement protein (MIP) is a lipoprotein, which is involved in the process of chlamydia infection and inflammation as an immune dominant antigen. In this study, MIP coding gene was amplified by PCR using the published sequence of GenBank. The gene was digested and ligated by enzyme. The expression vector pET-mip was successfully constructed and transformed into E. coli BL21 (DE3). SDS-PAGE analysis showed that there were specific bands at 27kD, and pET-mip was transformed into E. coli BL21 (DE3). The results showed that the inserted mip gene was successfully expressed by Western-blot assay. The purified MIP protein was labeled with HRP as the enzyme labeled antigen, the optimal coating concentration was 2.5 渭 g / mL, and the optimal dilution of serum was 1: 100. A double antigen sandwich Elisa was established. Compared with IHA, the established double-antigen Elisa had a higher sensitivity and a coincidence rate of 89.3%, and had no cross reaction with mycoplasma, Legionella pneumophila, foot-and-mouth disease and so on. High sensitivity and good repeatability. After immunizing Balb / c mice with purified MIP protein, two hybridoma cell lines were established, which secreted monoclonal antibodies against MIP protein stably, and were named M1 and M3, respectively. The titer of monoclonal antibody supernatant was 1: 1600 by indirect Elisa, and chromosome identification was consistent with the characteristics of hybridoma cells. The maximum binding concentration of the two McAbs was 10 渭 g / mL Western-blot. The two monoclonal antibodies could specifically recognize MIP protein mouse monoclonal antibody subclass detection kit. The two McAb immunoglobulin types were IgG1 and the light chain was 魏 chain. Chlamydia infection was effectively neutralized by two McAbs inoculated with chicken embryo. In conclusion, this study successfully expressed the MIP protein of Chlamydia abortus, established a sandwich Elisa method for the diagnosis of MIP protein, and prepared anti-MIP monoclonal antibody. It lays a foundation for the accurate diagnosis of chlamydia miscarriage and the study of related pathogenic mechanism.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.67

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