發(fā)布時(shí)間：2018-07-03 12:45

  本文選題：型鴨甲肝病毒 + VP蛋白��； 參考：《畜牧獸醫(yī)學(xué)報(bào)》2016年01期

【摘要】：旨在探究1型鴨甲肝病毒(DHAV-1)VP3蛋白抗血清的中和活性并鑒定VP3的線性B細(xì)胞表位。利用pGEX-4T-1表達(dá)載體,在BL21(DE3)宿主菌中原核表達(dá)DHAV-1 VP3基因,以切膠純化出的蛋白質(zhì)為抗原免疫兔制備多克隆抗體,通過(guò)雞胚中和試驗(yàn)對(duì)多抗的中和效價(jià)進(jìn)行檢測(cè);采用KarplusSchulz、Emini、Jameson-Wolf和Parker方法分別對(duì)柔韌性、表面可及性、抗原性及親水性進(jìn)行分析,得到了4條候選線性B細(xì)胞表位,以制備的兔抗VP3多克隆抗體為一抗,通過(guò)間接ELISA方法對(duì)人工合成的B細(xì)胞表位進(jìn)行鑒定,并進(jìn)一步用臨床鴨血清樣品對(duì)鑒定的B細(xì)胞表位的抗體檢測(cè)能力進(jìn)行評(píng)估。結(jié)果顯示,VP3在BL21(DE3)中以包涵體形式表達(dá),大小約54ku,Western blot分析表明重組蛋白質(zhì)具有較好的反應(yīng)原性。制備的兔抗VP3多克隆抗體的瓊擴(kuò)效價(jià)達(dá)到1∶16,并能中和DHAV-1,中和效價(jià)為1∶39;利用間接ELISA鑒定出GKRKPCRRPIHKPKNPPQEP(1—20aa)、FNTGRYQMSWYPIADGEQSL(131—150aa)和VNSSAPSNID(200—209aa)為VP3的B細(xì)胞表位,抗體檢測(cè)能力試驗(yàn)結(jié)果顯示表位肽可檢測(cè)臨床DHAV-1鴨血清。本研究表明DHAV-1VP3的抗血清具備一定的中和活性,1—20aa、131—150aa和200—209aa為VP3的B細(xì)胞表位且具有臨床應(yīng)用前景。
[Abstract]:The aim of this study was to investigate the neutralization activity of DHAV-1 VP3 antiserum and to identify the linear B cell epitopes of VP3. Using pGEX-4T-1 expression vector, DHAV-1 VP3 gene was expressed in BL21 (DE3) host strain. The polyclonal antibody was prepared by immunizing rabbits with the purified protein. The neutralization titer of the polyclonal antibody was detected by chicken embryo neutralization test. The flexibility, surface accessibility, antigenicity and hydrophilicity of four candidate linear B cell epitopes were analyzed by Karplus Schulzian minieson-Wolf and Parker methods respectively. The prepared rabbit anti-VP3 polyclonal antibody was used as the first antibody. The synthetic B cell epitopes were identified by indirect Elisa, and the antibody detection ability of the identified B cell epitopes was further evaluated with clinical duck serum samples. The results showed that VP3 was expressed as inclusion body in BL21 (DE3). Western blot analysis showed that the recombinant protein had good reactivity. The agarose expansion titer of rabbit anti-VP3 polyclonal antibody was 1: 16, and the neutralization titer of DHAV-1 was 1: 39. GKRKPCRPIHKPKNPPQEP (1-20aa) FNTGRYQMSWYPIADGEQSL (131-150aa) and VNSSAPSNID (200-209aa) were identified as the epitopes of VP3. The results of antibody test showed that epitope peptide could detect clinical DHAV-1 duck serum. The results showed that the antiserum of DHAV-1 VP3 had certain neutralization activity and the B cell epitopes of DHAV-1VP3 and 200-209aa were B cell epitopes of VP3 and had the prospect of clinical application.
【作者單位】： 四川農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院禽病防治研究中心;四川農(nóng)業(yè)大學(xué)預(yù)防獸醫(yī)研究所;動(dòng)物疫病與人類(lèi)健康四川省重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家“十二五”科技支撐計(jì)劃(2015BAD12B05) 國(guó)家現(xiàn)代農(nóng)業(yè)(水禽)產(chǎn)業(yè)技術(shù)體系專(zhuān)項(xiàng)(CARS-43-8) 四川省創(chuàng)新團(tuán)隊(duì)項(xiàng)目(12TD005/2013TD0015)
【分類(lèi)號(hào)】：S858.32

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