噬菌体展示技术筛选PCV2 Cap蛋白的抗原表位

发布时间：2018-05-15 17:52

本文选题：猪圆环病毒2 + Cap蛋白　；参考：《湖南农业大学》2012年硕士论文

【摘要】：为详细绘制PCV2Cap蛋白的抗原表位,在本研究中我们利用噬菌体展示技术对亲和纯化的Cap特异抗体识别的表位进行了筛选及鉴定,主要工作如下：首先,利用硫酸铵盐析法从收集的PCV2阳性血清中提取总IgG；大量表达重组Cap蛋白并进行纯化浓缩,将浓缩后的Cap蛋白与溴化氰活化琼脂糖凝胶4B偶联,制备亲和层析柱；再利用该柱子从总IgG中纯化抗Cap蛋白特异抗体。结果显示1ml原始血清可获得约0.2mg特异抗体。其次,用PCV2Cap蛋白特异抗体筛选噬菌体随机7肽库,3轮筛选后,随机挑取27个噬斑进行ELISA检测及测序分析,结果显示有20个呈阳性反应,将该20个噬菌体所携带的7肽序列与Cap序列进行比对发现,此20个噬菌体表位对应于Cap蛋白的八个区域,所对应的Cap序列依次为53FGYT56,65PSW67,69VDMMR73,79FLPPG84,86SNPRSVPF93,103KVEFWP108,119GSSXXXLDD127和230PLNP233。与Cap表位相关的之前研究相比,8个肽段中有3个(53FGYT56,86SNPRSVPF93和103KVEFWP108)为本研究第一次鉴定出的,另外5个位于已有报道范围内(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个氨基酸组成,因而推断筛选出的肽段为Cap主要免疫区的核心序列,结合已有报道,可推出Cap蛋白47-127位氨基酸是一个多表位集中的区域。第三,选取代表上述八个区域的噬菌体各一个进行Western Blot分析,结果显示所选噬菌体均可与PCV2阳性血清反应,表明筛选到的噬菌体表位具有反应原性。另将上述八个噬菌体进行小鼠免疫试验,发现携带有Cap核心序列53FGYT56和230PLNP233的噬菌体不能诱导小鼠产生相应的免疫应答,而其余各组均可诱导抗体的产生。综上,本研究用亲和纯化的PCV2Cap特异抗体筛选噬菌体随机7肽库,共获得8个拟表位(5个位于已有报道范围内,3个为新发现的),对此8个拟表位进行反应原性及免疫原性分析,确定了Cap蛋白两个新的表位,(?)(?)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.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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