本文選題：1型鴨甲肝病毒　切入點(diǎn)：VP3蛋白　出處：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：鴨甲型肝炎病毒(duck hepatitis A virus, DHAV)是一種無(wú)囊膜的單股正鏈RNA病毒,基因組RNA編碼結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白。其中,結(jié)構(gòu)蛋白對(duì)病毒的組裝和致病性有重要作用。VP3蛋白是主要的結(jié)構(gòu)蛋白,位于衣殼表面,相對(duì)保守,是病毒粒子抗原性的主要決定因子,存在著眾多抗原表位,可誘導(dǎo)機(jī)體產(chǎn)生免疫應(yīng)答。1型鴨甲肝病毒(DHAV-1)是分布最廣、危害最大的DHAV,本研究對(duì)DHAV-1的VP3進(jìn)行原核表達(dá)和純化,并制備兔抗VP3多克隆抗體,通過(guò)雞胚中和試驗(yàn)探究VP3多抗血清的中和活性；通過(guò)間接ELISA對(duì)VP3上可能存在的B抗原表位進(jìn)行鑒定；建立基于VP3的檢測(cè)DHAV抗體的間接ELISA方法。1. DHAV-1 VP3的原核表達(dá)、純化及鑒定通過(guò)RT-PCR得到了預(yù)期大小的VP3片段,將目的片段先后T-A克隆至pMD19-T (simple)和亞克隆至pGEX-4T-1,分別構(gòu)建了重組T質(zhì)粒pMD19-VP3和重組表達(dá)質(zhì)粒pGEX-VP3。 pGEX-VP3轉(zhuǎn)化BL21(DE3)后表達(dá)的重組蛋白約50kD。以0.2mmol/LIPTG、37℃誘導(dǎo)8h表達(dá)量最大,且始終以包涵體形式表達(dá)。采用切膠純化獲得的重組蛋白純度高。Western blot表明重組蛋白可被兔抗DHAV-1抗體識(shí)別,具有良好的反應(yīng)原性。2. DHAV-1 VP3的抗血清中和活性分析及B細(xì)胞表位鑒定利用純化的VP3重組蛋白乳劑免疫雄兔,經(jīng)五免后瓊脂擴(kuò)散試驗(yàn)的效價(jià)均達(dá)到1：16。Western blot證明兔抗血清可與VP3蛋白特異性結(jié)合。雞胚中和試驗(yàn)表明VP3抗血清的中和效價(jià)為2-5.29。運(yùn)用生物信息學(xué)軟件綜合分析VP3序列的柔韌性、表面可及性、親水性和抗原性,預(yù)測(cè)出四條B細(xì)胞表位多肽送公司合成。以制備的VP3多克隆抗體作一抗,VP3蛋白作抗原進(jìn)行間接ELISA,確定了表位肽的最佳稀釋度為1：1280,然后以該濃度稀釋的表位肽作抗原進(jìn)行間接ELISA鑒定B細(xì)胞表位,結(jié)果證明1GKRKPCRRPIHKPKNPPQEP20、 131FNTGRYQMSWYPIADGEQSL150和200VNSSAPSNID209能與VP3多克隆抗體特異性結(jié)合,為VP3的B細(xì)胞表位。進(jìn)一步用DHAV-1臨床鴨血清樣品對(duì)B表位的抗體檢測(cè)能力進(jìn)行評(píng)估,結(jié)果表明表位肽可用于檢測(cè)DHAV-1抗體。3.基于DHAV-1 VP3蛋白的間接ELISA方法的建立通過(guò)條件的摸索,得到以VP3作為包被抗原的間接ELISA方法的最佳檢測(cè)條件為：以9.375 ng/μL蛋白濃度4℃包被過(guò)夜,1%明膠37±封閉90min,加1：160稀釋的被檢血清于37±孵育90min,1：2000稀釋的HRP標(biāo)記兔抗鴨IgG于37±孵育45min。確定了陽(yáng)性閾值為0.332。該方法有良好的敏感性、重復(fù)性及特異性,并能同時(shí)檢測(cè)出DHAV-1和DHAV-3的抗體,與DHAV-1全病毒作抗原的間接ELISA方法的符合率為96%。
[Abstract]:Duck hepatitis A virus (DHAV) is a single-stranded positive strand RNA virus with no envelope. The genomic RNA encodes structural and non-structural proteins. Structural proteins play an important role in the assembly and pathogenicity of virus. VP3 protein is the main structural protein, which is located on the surface of the capsid and is relatively conservative. It is the main determinant of the antigenicity of virus particles and has many antigenic epitopes. DHAV-1 is one of the most widely distributed and harmful duck hepatitis A virus (DHAV-1). In this study, the VP3 of DHAV-1 was expressed and purified in prokaryotic, and rabbit polyclonal antibody against VP3 was prepared. The neutralization activity of VP3 polyantiserum was studied by chicken embryo neutralization test, the possible B antigen epitopes on VP3 were identified by indirect ELISA, and the indirect ELISA method based on VP3 was established to detect DHAV antibody. 1. Prokaryotic expression of DHAV-1 VP3. The expected size of VP3 fragment was obtained by RT-PCR. After T-A was cloned into pMD19-T simpleand subcloned into pGEX-4T-1, the recombinant T plasmid pMD19-VP3 and the recombinant expression plasmid pGEX-VP3. pGEX-VP3 were transformed into BL21DE3, respectively. The recombinant protein expressed at 0.2 mmol / L LIPTGN 37 鈩，

本文編號(hào)：1570358