本文選題：細粒棘球絳蟲 + EdiagA864��； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：細粒棘球絳蟲(Echinococcus granulosus)寄生在犬的小腸內(nèi)，其感染性蟲卵或者分泌的節(jié)片可隨犬糞便排出體外，造成人和家畜的感染，嚴重威脅人類健康和畜牧業(yè)的快速發(fā)展，給世界經(jīng)濟造成了嚴重的損失。因此，此病的防治對人類健康和畜牧業(yè)發(fā)展非常重要。一直以來，各國研究者們努力探索用來快速診斷、控制并預(yù)防該病的有效措施及方法，雖然獲得了很大的突破，但卻從未從根本上阻止該病的流行。犬是該蟲的唯一終末宿主，因此犬細粒棘球絳蟲的防治對控制此病的流行具有重要意義。目前，細粒棘球絳蟲糞抗原的檢測方法主要有檳榔堿導(dǎo)瀉法、ELISA方法、PCR方法等，但是檳榔堿導(dǎo)瀉法的反應(yīng)率具有一定的局限性；ELISA方法常出現(xiàn)敏感性低，并伴有一定的交叉反應(yīng)；PCR方法操作復(fù)雜，并對儀器要求較高。這些方法檢測水平常常參差不齊，所用檢測抗原也是包括成蟲蟲體抗原、囊液抗原、原頭節(jié)蟲體抗原及其分泌物抗原等許多種類。但至今還沒有檢測犬細粒棘球絳蟲的金標準方法和抗原。因此，選擇高特異性的抗原，建立具有高敏感性、高特異性的診斷方法十分必要。本研究利用抗細粒棘球絳蟲高免血清，篩選出細粒棘球絳蟲的免疫相關(guān)抗原，經(jīng)原核表達后制備了單克隆抗體和多克隆抗體，建立雙抗夾心ELISA方法檢測犬糞便抗原，為尋找有效的診斷抗原和建立高效、特異的診斷方法奠定了基礎(chǔ)。 細粒棘球絳蟲相關(guān)抗原基因的篩選及原核表達本試驗采用SEREX技術(shù)對細粒棘球絳蟲cDNA文庫進行相關(guān)抗原篩選。對文庫λ噬菌體進行平板培養(yǎng)，用IPTG誘導(dǎo)其表達蛋白，用兔抗細粒棘球絳蟲成蟲高免血清、HRP標記的羊抗兔IgG對所表達的蛋白進行陽性篩選，共獲得149個陽性噬菌斑，，后對其插入片段進行PCR擴增和序列分析，得到與絳蟲屬相關(guān)的基因序列10個。其中，具有864bp的開放性閱讀框的序列，編碼288個氨基酸，經(jīng)NCBI BLAST氨基酸同源性分析與多房棘球絳蟲診斷抗原gp50的氨基酸序列（CDJ06164.1）有92%同源性，是一個未知的新基因，本研究將其命名為：EdiagA864。對其成功構(gòu)建克隆載體和表達載體，經(jīng)原核表達得到大小約為51kDa的重組蛋白，經(jīng)Western blot分析其具有良好的反應(yīng)原性。 EdiagA864蛋白單克隆抗體的制備與純化以EdiagA864蛋白為抗原，經(jīng)免疫Balb/c小鼠、細胞融合以及雜交瘤篩選與克隆，制備了共四株抗EdiagA864蛋白的單克隆抗體，分別為2D12、3H4、4A6和6E5。經(jīng)過染色體分析及抗體亞型的鑒定，得到2D12、3H4、4A6和6E5的細胞染色體數(shù)分別為97、101、98、96。4株單克隆抗體的重鏈亞型為為IgG1，輕鏈亞型為Kappa。2D12、3H4腹水效價為20萬，4A6和6E5腹水效價為10萬。經(jīng)純化后可見一條輕鏈與一條重鏈。 EdiagA864蛋白多克隆抗體的制備與純化以EdiagA864蛋白為抗原，經(jīng)免疫日本大耳白兔，成功制備了兔抗EdiagA864多克隆抗體，經(jīng)過Western blot驗證，該抗體能夠特異性識別EdiagA864蛋白，并與弓形蟲、新孢子蟲和犬心絲蟲陽性血清均無交叉反應(yīng)。 犬細粒棘球絳蟲雙抗體夾心ELISA方法的建立本試驗利用制備的單克隆抗體與兔多克隆抗體建立了三種雙抗夾心ELISA方法：一是以單克隆抗體2D12作為捕獲抗體，HRP標記的多克隆抗體作為檢測抗體；二是以2D12和3H4兩種單克隆抗體混合作為捕獲抗體，HRP標記的多克隆抗體作為檢測抗體；三是以單克隆抗體3H4作為捕獲抗體，HRP標記的單克隆抗體2D12作為檢測抗體。特異性試驗中，三種方法與賈第蟲陽性犬糞便樣品、蛔蟲陽性犬糞便樣品均無交叉反應(yīng)；敏感性試驗中，方法一和方法二的敏感性為標準陽性樣本稀釋至1:20，方法三的敏感性為標準陽性樣本稀釋至1:5。應(yīng)用三種方法對細粒棘球絳蟲陽性犬糞便樣品進行了檢測，方法一和方法二陽性檢出率為100%（8/8），方法三的陽性檢出率為87.5%（7/8）。應(yīng)用三種雙抗夾心ELISA方法檢測64份來自長春地區(qū)的待檢樣品，結(jié)果均為陰性。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.9

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