本文選題：豬圓環(huán)病毒2 + Cap蛋白��； 參考：《湖南農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：為詳細(xì)繪制PCV2Cap蛋白的抗原表位,在本研究中我們利用噬菌體展示技術(shù)對親和純化的Cap特異抗體識別的表位進(jìn)行了篩選及鑒定,主要工作如下： 首先,利用硫酸銨鹽析法從收集的PCV2陽性血清中提取總IgG；大量表達(dá)重組Cap蛋白并進(jìn)行純化濃縮,將濃縮后的Cap蛋白與溴化氰活化瓊脂糖凝膠4B偶聯(lián),制備親和層析柱；再利用該柱子從總IgG中純化抗Cap蛋白特異抗體。結(jié)果顯示1ml原始血清可獲得約0.2mg特異抗體。 其次,用PCV2Cap蛋白特異抗體篩選噬菌體隨機7肽庫,3輪篩選后,隨機挑取27個噬斑進(jìn)行ELISA檢測及測序分析,結(jié)果顯示有20個呈陽性反應(yīng),將該20個噬菌體所攜帶的7肽序列與Cap序列進(jìn)行比對發(fā)現(xiàn),此20個噬菌體表位對應(yīng)于Cap蛋白的八個區(qū)域,所對應(yīng)的Cap序列依次為53FGYT56,65PSW67,69VDMMR73,79FLPPG84,86SNPRSVPF93,103KVEFWP108,119GSSXXXLDD127和230PLNP233。與Cap表位相關(guān)的之前研究相比,8個肽段中有3個(53FGYT56,86SNPRSVPF93和103KVEFWP108)為本研究第一次鑒定出的,另外5個位于已有報道范圍內(nèi)(65PSW67,69VDMMR73,79FLPPG84位于65-85aa;119GSSXXXLDD127位于117-131aa;230PLNP233位于表位231-233),抗原表位一般由6-8個氨基酸組成,因而推斷篩選出的肽段為Cap主要免疫區(qū)的核心序列,結(jié)合已有報道,可推出Cap蛋白47-127位氨基酸是一個多表位集中的區(qū)域。 第三,選取代表上述八個區(qū)域的噬菌體各一個進(jìn)行Western Blot分析,結(jié)果顯示所選噬菌體均可與PCV2陽性血清反應(yīng),表明篩選到的噬菌體表位具有反應(yīng)原性。另將上述八個噬菌體進(jìn)行小鼠免疫試驗,發(fā)現(xiàn)攜帶有Cap核心序列53FGYT56和230PLNP233的噬菌體不能誘導(dǎo)小鼠產(chǎn)生相應(yīng)的免疫應(yīng)答,而其余各組均可誘導(dǎo)抗體的產(chǎn)生。 綜上,本研究用親和純化的PCV2Cap特異抗體篩選噬菌體隨機7肽庫,共獲得8個擬表位(5個位于已有報道范圍內(nèi),3個為新發(fā)現(xiàn)的),對此8個擬表位進(jìn)行反應(yīng)原性及免疫原性分析,確定了Cap蛋白兩個新的表位,(?)(?)86SNPRSVPF93和103KVEFWP108。
[Abstract]:In order to map the epitopes of PCV2Cap protein in detail, we used phage display technique to screen and identify the epitopes recognized by affinity purified Cap specific antibodies. The main work is as follows: Firstly, total PCV2 was extracted from collected PCV2 positive serum by ammonium sulfate salting-out method, the recombinant Cap protein was expressed in large quantities and purified and concentrated, the concentrated Cap protein was coupled with activated agarose gel 4B by cyanide bromide, and the affinity chromatography column was prepared. The specific antibody against Cap protein was purified from total IgG by this column. The results showed that about 0.2mg specific antibody could be obtained from the original serum of 1ml. Secondly, after 3 rounds of phage screening with specific antibodies to PCV2Cap protein, 27 phages were randomly selected for ELISA detection and sequencing analysis. The results showed that 20 phages were positive. The seven peptide sequences carried by the 20 phages were compared with the Cap sequences. The 20 phage epitopes corresponded to the eight regions of the Cap protein, and the corresponding Cap sequences were 53FGYT56n65PSW679 FLPPG84n86SNPRSVPF93103KVEFWP1081GSSXXXLDD127 and 230PLNP233. Compared with previous studies related to Cap epitopes, three of the 8 peptides were identified for the first time by this study. The other five peptides were located in the reported region of 65PSW67n69VDMMR7379FLPPG84 at 65-85aa1a 119GSSXXXLDD127 at 117-131aaPNP233 at epitope 231-233N, and the epitopes were generally composed of 6-8 amino acids. Therefore, it is inferred that the selected peptide is the core sequence of the main immune region of Cap. Combined with the previous reports, it can be deduced that the amino acid of 47-127 amino acids of Cap protein is a multi-epitope set region. Third, one phage representing the above eight regions was selected for Western Blot analysis. The results showed that the selected phage could react with the PCV2 positive serum, indicating that the selected phage epitope was reactive. In addition, the above eight phages were tested in mice. It was found that the phage carrying Cap core sequences 53FGYT56 and 230PLNP233 could not induce the corresponding immune response in mice, but the other groups could induce the production of antibodies. In this study, the phage random 7 peptide library was screened by affinity purified PCV2Cap specific antibody. Eight epitopes (5 were located in the reported area and 3 were newly discovered) were obtained. The reactivity and immunogenicity of the 8 epitopes were analyzed. Two new epitopes of Cap protein were identified, I. e. 86 SNPRSVPF93 and 103 KVEFWP108.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 姚寧;姚倫廣;張詳滿;郭泰林;闞云超;;噬菌體展示技術(shù)篩選抗輪狀病毒多肽的試驗研究[J];生物工程學(xué)報;2007年03期

2 吳健敏,任兆鈞,余興龍,涂長春,張念祖;利用T4噬菌體展示豬瘟病毒E2抗原[J];中國生物工程雜志;2004年11期

3 安燁;王宏俊;張培君;;利用噬菌體展示肽庫篩選雞傳染性支氣管炎病毒模擬抗原表位[J];生物技術(shù)通訊;2007年06期

4 王長軍,唐家琪,陶開華,李先富,白薇,郭恒彬;應(yīng)用噬菌體展示技術(shù)篩選、鑒定抗?jié)h灘病毒單克隆抗體的模擬表位[J];細(xì)胞與分子免疫學(xué)雜志;2004年04期

5 趙東升;劉有昌;安福生;李祥健;;近年來我國豬圓環(huán)病毒病的流行狀況及分析[J];養(yǎng)豬;2009年01期

6 郎洪武,張廣川,吳發(fā)權(quán),張創(chuàng)貴;斷奶豬多系統(tǒng)衰弱綜合征血清抗體檢測[J];中國獸醫(yī)科技;2000年03期

7 劉光清,倪征,云濤,梁華麗,華炯剛,李雙茂,杜清云;豬圓環(huán)病毒II型結(jié)構(gòu)蛋白的二級結(jié)構(gòu)及其B細(xì)胞抗原表位預(yù)測[J];中國預(yù)防獸醫(yī)學(xué)報;2005年06期

8 苗麗娟;符芳;王小武;陳微晶;魯晶紅;李曦;;豬圓環(huán)病毒2型去核定位信號ORF2基因的原核表達(dá)與初步應(yīng)用[J];中國預(yù)防獸醫(yī)學(xué)報;2008年05期

9 于永忠;李集臨;徐香玲;于力;;噬菌體展示技術(shù)與病毒抗原表位研究[J];中國預(yù)防獸醫(yī)學(xué)報;2009年03期

10 郭龍軍;陸月華;危艷武;黃立平;劉長明;;我國部分地區(qū)豬圓環(huán)病毒2型分離株的遺傳變異分析[J];中國預(yù)防獸醫(yī)學(xué)報;2009年11期

，

本文編號：1893321