本文選題：水貂阿留申病 + B細(xì)胞表位　； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：水貂阿留申�。ˋleutian disease，AD）是由水貂阿留申病毒（Aleutian diseasevirus，ADV）引起的自身免疫性、傳染性疾病，水貂感染ADV產(chǎn)生的抗體不但不能有效清除病毒，反而通過(guò)Fc受體介導(dǎo)的抗體依賴性增強(qiáng)作用（AntibodyDependent Enhancement, ADE）使病毒感染增強(qiáng)，導(dǎo)致水貂免疫系統(tǒng)功能紊亂。該病不僅導(dǎo)致貂群的繁殖能力下降，，而且在一定程度上能引起水貂死亡，給養(yǎng)貂業(yè)帶來(lái)了巨大的經(jīng)濟(jì)損失，也嚴(yán)重阻礙我國(guó)毛皮動(dòng)物養(yǎng)殖業(yè)發(fā)展。目前尚無(wú)有效疫苗治療該病，因此國(guó)內(nèi)外主要通過(guò)抗體檢測(cè)技術(shù)如對(duì)流免疫電泳（Counterimmune electrophoresis，CIEP）、酶聯(lián)免疫吸附實(shí)驗(yàn)（Enzyme-linked immunosorbentassay，ELISA）篩選并淘汰陽(yáng)性水貂，從而控制和凈化該病。 本研究目的在于建立有效的水貂阿留申病間接ELISA檢測(cè)方法。以水貂阿留申病美國(guó)株ADV-G結(jié)構(gòu)蛋白VP2為主要參考，通過(guò)B細(xì)胞抗原表位預(yù)測(cè)和保守序列篩選，獲得保守性高的優(yōu)勢(shì)B細(xì)胞抗原表位SD序列。通過(guò)大腸桿菌表達(dá)系統(tǒng)誘導(dǎo)表達(dá)，獲得了可溶性SD蛋白，大小為20KDa。表達(dá)產(chǎn)物經(jīng)鎳柱純化、超濾管濃縮后獲得高純度、高濃度的SD蛋白。Western Blot分析結(jié)果顯示，SD蛋白具有良好的反應(yīng)原性。將該蛋白作為CIEP檢測(cè)抗原與商業(yè)CIEP抗原蛋白同時(shí)檢測(cè)水貂血清，達(dá)到一致的檢測(cè)結(jié)果。 將獲得的抗原SD蛋白和三氨丙基三乙氧基硅烷（3-Aminopropyltriethoxysilane，APTES）共同包被建立間接ELISA檢測(cè)方法。通過(guò)方陣滴定法，摸索抗原SD蛋白和APTES的包被濃度，并摸索了包被和封閉時(shí)間，以及一抗和二抗的稀釋度，最終確定包被的抗原SD蛋白和APTES的濃度分別為10ug/mL和1%，一抗稀釋度為1:100，二抗稀釋度為1:3000，其中與傳統(tǒng)間接ELISA比較，包被時(shí)間、封閉時(shí)間均縮短為30min。特異性試驗(yàn)表明建立的該方法的SD蛋白僅能被水貂阿留申病毒抗體識(shí)別，而與犬細(xì)小病毒、偽狂犬病毒、犬瘟熱病毒、水貂腸炎病毒的陽(yáng)性血清和水貂阿留申病陰性血清均無(wú)交叉反應(yīng)；敏感性試驗(yàn)表明建立的該方法的敏感性高于CIEP；重復(fù)性試驗(yàn)表明批內(nèi)重復(fù)性和批間重復(fù)性的變異系數(shù)分別為0.612%~5.956%和0.135%~7.614%，均小于10%；臨床樣品檢測(cè)結(jié)果與CIEP檢測(cè)結(jié)果的符合率為93.2%。通過(guò)以上實(shí)驗(yàn)得出結(jié)論：本實(shí)驗(yàn)中通過(guò)設(shè)計(jì)獲得的全新的人工抗原SD蛋白具有良好的反應(yīng)原性和作為檢測(cè)抗原的能力。將SD蛋白作為包被抗原蛋白建立的間接ELISA檢測(cè)方法具有特異性好、敏感性高、重復(fù)性好、高效快捷的特點(diǎn)，且與CIEP檢測(cè)方法比較具有較高的符合率。本研究不僅為常用檢測(cè)方法CIEP提供更合適的基因工程抗原蛋白，也為開(kāi)發(fā)檢測(cè)水貂阿留申病的ELISA試劑盒及全自動(dòng)ELISA檢測(cè)方法提供基礎(chǔ)研究。
[Abstract]:Aleutian disease of mink (Aleutian disease ADV) is caused by Aleutian disease virus of mink (Aleutian disease virus ADV).On the contrary, Antibody Dependent Enhancement (ADE), mediated by FC receptor, enhanced the virus infection and resulted in the disorder of the immune system of mink.The disease not only leads to the decline of the breeding ability of mink, but also can cause the death of mink to a certain extent, which brings huge economic loss to mink industry, and seriously hinders the development of fur animal breeding industry in China.At present, there is no effective vaccine for the treatment of the disease, so antibody detection techniques such as counter immune electrophoresis and Enzyme-linked immunosorbent enzyme linked immunosorbent assay (Elisa) are used to screen and eliminate positive minks in order to control and purify the disease.The aim of this study was to establish an effective indirect ELISA method for detection of Aleutian disease in mink.The dominant B cell epitope SD sequence was obtained by predication of B cell epitopes and screening of conserved sequences with reference to the ADV-G structural protein VP2 of Aleutian disease American strain of mink.The expression of soluble SD protein was induced by Escherichia coli expression system and the size of SD protein was 20 K Da.The expressed product was purified by nickel column and concentrated in ultrafiltration tube to obtain high purity. Western Blot analysis of high concentration of SD protein showed that SD protein had good reactivity.Using this protein as CIEP detection antigen and commercial CIEP antigen protein, the mink serum was detected simultaneously, and the results were consistent.An indirect ELISA method was established by co-coating the obtained antigen SD protein with triaminopropyltriethoxysilane trisilane 3-Aminopropyltriethoxysilaneus (APTES).The encapsulation concentration of antigenic SD protein and APTES, the encapsulation and blocking time, and the dilution of the first antibody and the second antibody were studied by the method of square array titration.Finally, the concentration of antigen SD protein and APTES were determined to be 10ug/mL and 1, respectively. The dilution of the first antibody was 1: 100, and the dilution of the second antibody was 1: 3000. Compared with the traditional indirect ELISA, the encapsulation time and the blocking time were shortened to 30 mins.The specific test showed that the SD protein of this method can only be recognized by mink Aleutian virus antibody, but with canine parvovirus, pseudorabies virus, canine distemper virus,There was no cross reaction between the positive serum of mink enteritis virus and the negative serum of mink Aleutin disease.The sensitivity test showed that the sensitivity of this method was higher than that of CIEP, the coefficient of variation of repeatability within and between batches was 0.6125.956% and 0.135% 7.614, respectively, and the coincidence rate between the results of clinical samples and that of CIEP was 93. 2%.It is concluded from the above experiments that the new artificial antigen SD protein obtained in this experiment has good reactivity and ability to detect antigens.The indirect ELISA detection method using SD protein as coated antigen protein has the characteristics of good specificity, high sensitivity, good reproducibility, high efficiency and rapidity, and has a higher coincidence rate compared with CIEP detection method.This study not only provides a more suitable genetic engineering antigen protein for common detection method CIEP, but also provides basic research for the development of ELISA kit and automatic ELISA detection method for Aleutian disease in mink.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.92

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