犬细粒棘球绦虫EdiagA864单克隆抗体的制备及初步应用

发布时间：2018-07-11 09:57

本文选题：细粒棘球绦虫 + EdiagA864　；参考：《吉林大学》2015年硕士论文

【摘要】：细粒棘球绦虫(Echinococcus granulosus)寄生在犬的小肠内，其感染性虫卵或者分泌的节片可随犬粪便排出体外，造成人和家畜的感染，严重威胁人类健康和畜牧业的快速发展，给世界经济造成了严重的损失。因此，此病的防治对人类健康和畜牧业发展非常重要。一直以来，各国研究者们努力探索用来快速诊断、控制并预防该病的有效措施及方法，虽然获得了很大的突破，但却从未从根本上阻止该病的流行。犬是该虫的唯一终末宿主，因此犬细粒棘球绦虫的防治对控制此病的流行具有重要意义。目前，细粒棘球绦虫粪抗原的检测方法主要有槟榔碱导泻法、ELISA方法、PCR方法等，但是槟榔碱导泻法的反应率具有一定的局限性；ELISA方法常出现敏感性低，并伴有一定的交叉反应；PCR方法操作复杂，并对仪器要求较高。这些方法检测水平常常参差不齐，所用检测抗原也是包括成虫虫体抗原、囊液抗原、原头节虫体抗原及其分泌物抗原等许多种类。但至今还没有检测犬细粒棘球绦虫的金标准方法和抗原。因此，选择高特异性的抗原，建立具有高敏感性、高特异性的诊断方法十分必要。本研究利用抗细粒棘球绦虫高免血清，筛选出细粒棘球绦虫的免疫相关抗原，经原核表达后制备了单克隆抗体和多克隆抗体，建立双抗夹心ELISA方法检测犬粪便抗原，为寻找有效的诊断抗原和建立高效、特异的诊断方法奠定了基础。细粒棘球绦虫相关抗原基因的筛选及原核表达本试验采用SEREX技术对细粒棘球绦虫cDNA文库进行相关抗原筛选。对文库λ噬菌体进行平板培养，用IPTG诱导其表达蛋白，用兔抗细粒棘球绦虫成虫高免血清、HRP标记的羊抗兔IgG对所表达的蛋白进行阳性筛选，共获得149个阳性噬菌斑，，后对其插入片段进行PCR扩增和序列分析，得到与绦虫属相关的基因序列10个。其中，具有864bp的开放性阅读框的序列，编码288个氨基酸，经NCBI BLAST氨基酸同源性分析与多房棘球绦虫诊断抗原gp50的氨基酸序列（CDJ06164.1）有92%同源性，是一个未知的新基因，本研究将其命名为：EdiagA864。对其成功构建克隆载体和表达载体，经原核表达得到大小约为51kDa的重组蛋白，经Western blot分析其具有良好的反应原性。 EdiagA864蛋白单克隆抗体的制备与纯化以EdiagA864蛋白为抗原，经免疫Balb/c小鼠、细胞融合以及杂交瘤筛选与克隆，制备了共四株抗EdiagA864蛋白的单克隆抗体，分别为2D12、3H4、4A6和6E5。经过染色体分析及抗体亚型的鉴定，得到2D12、3H4、4A6和6E5的细胞染色体数分别为97、101、98、96。4株单克隆抗体的重链亚型为为IgG1，轻链亚型为Kappa。2D12、3H4腹水效价为20万，4A6和6E5腹水效价为10万。经纯化后可见一条轻链与一条重链。 EdiagA864蛋白多克隆抗体的制备与纯化以EdiagA864蛋白为抗原，经免疫日本大耳白兔，成功制备了兔抗EdiagA864多克隆抗体，经过Western blot验证，该抗体能够特异性识别EdiagA864蛋白，并与弓形虫、新孢子虫和犬心丝虫阳性血清均无交叉反应。犬细粒棘球绦虫双抗体夹心ELISA方法的建立本试验利用制备的单克隆抗体与兔多克隆抗体建立了三种双抗夹心ELISA方法：一是以单克隆抗体2D12作为捕获抗体，HRP标记的多克隆抗体作为检测抗体；二是以2D12和3H4两种单克隆抗体混合作为捕获抗体，HRP标记的多克隆抗体作为检测抗体；三是以单克隆抗体3H4作为捕获抗体，HRP标记的单克隆抗体2D12作为检测抗体。特异性试验中，三种方法与贾第虫阳性犬粪便样品、蛔虫阳性犬粪便样品均无交叉反应；敏感性试验中，方法一和方法二的敏感性为标准阳性样本稀释至1:20，方法三的敏感性为标准阳性样本稀释至1:5。应用三种方法对细粒棘球绦虫阳性犬粪便样品进行了检测，方法一和方法二阳性检出率为100%（8/8），方法三的阳性检出率为87.5%（7/8）。应用三种双抗夹心ELISA方法检测64份来自长春地区的待检样品，结果均为阴性。
[Abstract]:Echinococcus granulosus (Echinococcus granulosus) parasitic in the small intestine of the dog, its infective eggs or secreted segments can be discharged from the canine excrement, causing infection of human and livestock, seriously threatening the rapid development of human health and animal husbandry, causing severe losses to the world economy. Therefore, the prevention and control of this disease to human health and livestock The development of animal husbandry is very important. The researchers have been trying to explore the effective measures and methods to quickly diagnose, control and prevent the disease. Although a great breakthrough has been made, it has never fundamentally prevented the epidemic. The dog is the only terminal host of the worm, so the control of Echinococcus canis is to control the disease. The prevalence of Echinococcus granulosus is of great significance. At present, the methods of detecting the fecal antigen of Echinococcus granulosus are mainly arecoline catharsis, ELISA and PCR, but the reaction rate of arecoline purge method has some limitations; ELISA method often appears low sensitivity with a certain cross reaction; PCR method is complicated and requires more instruments. The detection level of these methods is often uneven, and the detection antigens are also including the adult worm body antigen, the cyst fluid antigen, the original somatic antigen and the secretion antigen, and so on. However, the gold standard method and antigen of Echinococcus canis have not been detected so far. Therefore, the high specific antigen is selected to establish the Gao Min sensibility. The high specificity of the diagnostic method is very necessary. In this study, the immuno related antigen of Echinococcus granulosus was screened using the high free serum of Echinococcus granulosus, and the monoclonal antibody and polyclonal antibody were prepared by the prokaryotic expression. The double anti sandwich ELISA method was established to detect the antigenic antigen of the dog feces, in order to find the effective diagnostic antigen and establish the high efficiency. The specific diagnostic method laid the foundation.
Screening and prokaryotic expression of Echinococcus granulosus related antigen gene, SEREX technique was used to screen the associated antigen of Echinococcus granulosus cDNA library. The library lambda bacteriophage was cultured, the expression protein was induced by IPTG, the Rabbit anti sera against Echinococcus granulosus adult, and the HRP labeled Sheep anti rabbit IgG on the egg 149 positive phagocytosis was obtained by positive screening. After PCR amplification and sequence analysis of the inserted fragments, 10 genes related to the genus Taenia were obtained. Among them, the sequence of the open reading frame with 864bp, encoding 288 amino acids, and the NCBI BLAST amino acid analysis and the diagnostic antigen GP50 of Echinococcus multiloculeus Amino acid sequence (CDJ06164.1) has 92% homology, which is an unknown new gene. This study named it as: EdiagA864. has successfully constructed cloning vector and expression vector. The recombinant protein is about 51kDa by prokaryotic expression, and it has good reactivity by Western blot.
The preparation and purification of EdiagA864 protein monoclonal antibody with EdiagA864 protein as antigen, immunized Balb/c mice, cell fusion and hybridoma screening and cloning were prepared, and four monoclonal antibodies against EdiagA864 protein were prepared. 2D12,3H4,4A6 and 6E5. were analyzed by chromosome analysis and identification of antibody subtypes, and 2D12,3H4,4A6 and 6E5 were obtained. The heavy chain subtypes of the cell chromosome number of 97101,98,96.4 strains were IgG1, the light chain subtype Kappa.2D12,3H4 ascites titer was 200 thousand, the 4A6 and 6E5 ascites titer was 100 thousand. After purification, a light chain and a heavy chain were found.
EdiagA864 protein polyclonal antibody was prepared and purified with EdiagA864 protein as an antigen. The Rabbit anti EdiagA864 polyclonal antibody was successfully prepared by immunizing Japanese rabbit. The antibody could identify EdiagA864 protein specifically by Western blot, and no cross reaction with Toxoplasma gondii, new spore worm and dog heart worm positive sera.
The establishment of a double antibody sandwich ELISA method for Echinococcus canine, three double anti sandwich ELISA methods were established by using the monoclonal antibody and rabbit polyclonal antibody. One was the monoclonal antibody 2D12 as the capture antibody, the HRP labeled polyclonal antibody was used as the detection antibody, and the two was mixed with two monoclonal antibodies of 2D12 and 3H4. As a capture antibody, the HRP labeled polyclonal antibody was used as a detection antibody; three was the monoclonal antibody 3H4 as the capture antibody and the HRP labeled monoclonal antibody 2D12 was used as the detection antibody. In the specificity test, the three methods were not cross reacted with the Giardia positive dog feces samples and the Ascaris positive dog feces samples. The sensitivity of the one and method two was diluted to the standard positive sample to 1:20, and the sensitivity of method three was diluted to the standard positive sample to 1:5. application. Three methods were used to detect the dog feces samples of Echinococcus granulosus. The positive rate of method one and method two was 100% (8/8), and the positive rate of method three was 87.5% (7/8). Three kinds of methods were applied. The double antibody sandwich ELISA method was used to detect 64 samples from Changchun. All the results were negative.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.9

【相似文献】