發(fā)布時間：2018-04-03 06:19

  本文選題：鴨腸炎病毒　切入點：糖蛋白B、C、D、E　出處：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：鴨病毒性腸炎(duck viral enteritis,DVE)是由鴨腸炎病毒(Duck enteritis virus,DEV)引起的一種急性、熱性、敗血性高度傳染性疾病,給水禽養(yǎng)殖造成巨大的經(jīng)濟損失。目前,常用的鴨病毒性腸炎抗體檢測方法中,中和試驗操作復(fù)雜,基于DEV的ELISA檢測病毒純化困難,成本較高,而以DEV某種囊膜糖蛋白抗原優(yōu)勢區(qū)建立的ELISA方法,檢測的抗體單一,因此需要一種操作簡單、成本低廉,且檢出抗體全面的的檢測方法。本研究旨在在篩選DEV囊膜蛋白B(g B)優(yōu)勢抗原區(qū)的基礎(chǔ)上,結(jié)合本實驗室已篩選的DEV g C、g D、g E的優(yōu)勢抗原表位,構(gòu)建基于DEV主要囊膜糖蛋白抗原優(yōu)勢區(qū)的復(fù)合表位,建立檢測DEV血清抗體的間接ELISA檢測方法。為深入解析DEV g B的主要抗原結(jié)構(gòu)及其特點,快速、簡便、特異、準確的檢測、監(jiān)測DEV抗體水平提供理論依據(jù)和技術(shù)支持。本研究通過重疊表位作圖法對DEV g B的B細胞線性表位進行篩選和鑒定,設(shè)計覆蓋g B整個區(qū)域的多肽片段,利用大腸桿菌p ET-32a表達系統(tǒng),實現(xiàn)了截短融合蛋白表達及純化,并結(jié)合Western-blot篩選出DEV g B的優(yōu)勢抗原區(qū),經(jīng)四輪篩選成功鑒定的兩個優(yōu)勢抗原區(qū),分別位于N端111-150aa(ATDRPHGLMNDQDTHLDGERLIRGVQSTREI)和506-530aa(QELTRDNRTELALDLLG AMRGDKTR),是現(xiàn)已知被鑒定的最小的優(yōu)勢抗原區(qū)。本研究通過間接ELISA方法對DEV的g B、g C、g D、g E截短融合蛋白的抗原性測定,進一步符合Western-blot的鑒定結(jié)果,同時對g B、g C、g D、g E抗原性強弱比較分析。研究發(fā)現(xiàn)間接ELISA檢測的結(jié)果與Western-blot基本吻合,且g B的抗原性最強,g C次之,g D稍弱,而g E最弱。本研究利用試驗DEV免疫的各周鴨血清對g B、g C、g D、g E抗體消長規(guī)律進行測定,結(jié)果顯示針對g B的抗體變化與針對DEV抗體基本一致,免疫后第一周即可檢測到特異性抗體,第三周達到峰值,而針對g C、g D和g E的抗體水平基本保持同步,免疫后第一周也可檢測到特異性抗體,第八周達到峰值。研究表明g B在免疫早期即表現(xiàn)出高的抗原性,而g C,g D、g E在免疫后期表現(xiàn)出高的抗原性,且g B抗原性明顯高于g C、g D、g E。通過對g B、g C、g D、gE抗原性強弱的比較,選擇抗原性最強的不同囊膜糖蛋白抗原優(yōu)勢區(qū)串聯(lián),設(shè)計合成了兩段復(fù)合表位DEV-1-GP和DEV-2-GP,基因優(yōu)化后以適應(yīng)大腸桿菌表達系統(tǒng),并利用同尾酶法反復(fù)串聯(lián)1~7拷貝構(gòu)建DEV-1、2-GP1~7,利用大腸桿菌p ET-30a表達系統(tǒng),實現(xiàn)了融合蛋白表達及純化,經(jīng)Western-blot和間接ELISA鑒定DEV-2-GP2蛋白表達量高,抗原性強,且為上清表達,可作為診斷抗原�；趶�(fù)合表位DEV-2-GP2的間接ELISA方法檢測血清中DEV抗體水平,確定間接ELISA檢測方法的臨界值為0.35,用建立的ELISA檢測方法對常見的幾種鴨病陽性血清:鴨病毒性肝炎、鴨源小鵝瘟、鴨減蛋綜合征、鴨源禽流感進行檢測,結(jié)果均為陰性,表明該ELISA檢測方法具有良好的特異性。最低檢出量為1:320,表明該方法敏感性強,批內(nèi)重復(fù)性試驗變異系數(shù)在1.50%~8.12%,批間重復(fù)性試驗變異系數(shù)在2.56%~8.89%,表明該方法具有良好的重復(fù)穩(wěn)定性。與OIE推薦的DEV抗體檢測中和試驗相比,符合率為82.8%,與市售的商品化試劑盒相比,符合率為76.7%,利用本方法可很好地監(jiān)測SPF鴨免疫鴨瘟弱毒疫苗的血清抗體變化,并對免疫鴨瘟弱毒疫苗的商品鴨及DEV免疫背景不清的現(xiàn)地鴨血清檢測,陽性率分別為50%和4.2%。說明本方法可初步應(yīng)用于鴨病毒性腸炎抗體的檢測,為我國鴨腸炎病毒的檢測和血清流行病學(xué)調(diào)查提供了有效的技術(shù)支持。
[Abstract]:Duck enteritis virus (duck viral, enteritis, DVE) by duck enteritis virus (Duck enteritis, virus, DEV) is an acute, heat caused by septic, highly contagious disease, causing huge economic losses to waterfowl breeding. At present, of duck enteritis virus antibody detection methods used in neutralization test complex detection of ELISA virus based on DEV purification difficult, high cost, ELISA method and DEV to a glycoprotein antigenic dominant region establishment, single antibody detection, so we need a simple operation, low cost, and the detection method for detecting antibody. The purpose of this study was to complete in the screening of DEV envelope protein B (g B) based dominant antigen regions, combined with the G C laboratory has screened DEV, G D, G E epitope construct composite table DEV glycoprotein antigen dominance region based on indirect ELIS, establish the detection of serum antibody against DEV A is the main antigen detection method. The structure and characteristics of DEV g B, in-depth analysis of the rapid, simple, specific, accurate detection, monitoring the antibody level of DEV and provide a theoretical basis and technical support. This study by overlapping epitope mapping method of DEV g B B cell linear epitopes were screened and identified, peptide design g B covering the entire region, the E.coli Expression System of P ET-32a, the truncated fusion protein expression and purification, and the combination of Western-blot DEV g were dominant antigen B, after four rounds of screening two dominant antigen regions successfully identified, respectively located in the N terminal 111-150aa (ATDRPHGLMNDQDTHLDGERLIRGVQSTREI) and 506-530aa (QELTRDNRTELALDLLG AMRGDKTR), is now known by the identification of minimal dominant antigen regions. This study by indirect ELISA method for DEV g B, G C, G D, G E truncated fusion protein antigenicity determination, in order to meet Western- blot鐨勯壌瀹氱粨鏋，

本文編號：1703974