鸭圆环病毒ORF3基因的克隆表达及其单克隆抗体的制备与应用

发布时间：2018-02-04 21:13

本文关键词： 鸭圆环病毒 ORF3基因克隆表达单克隆抗体间接竞争ELISA　出处：《四川农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：鸭圆环病毒被归类于圆环病毒科圆环病毒属,该病毒ORF3所编码的蛋白功能目前尚未阐释。为了制备针对该蛋白的单克隆抗体,给进一步探索该蛋白功能提供素材,论文对本实验室分离鉴定的DuCV ORF3基因开展了以下实验：DuCV ORF3基因的生物信息学分析、ORF3基因的克隆表达及纯化、单克隆抗体的制备、间接竞争ELISA方法的建立和应用,结果如下：1. DuCV ORF3的生物信息学分析GH01株DuCV全长1988 by,ORF3基因A、T、G、C含量分别为21.89%、25.25%、24.58%、28.28%,共297bp,位于全基因组第399至第103位碱基,编码98个氨基酸,分子量约11.6 KD。该蛋白二级结构中无规则卷曲占大部分,含量约为62.3%,α-螺旋和p-折叠结构分别占31.6%和6.1%；没有保守区、信号肽、跨膜区,提示该蛋白可能是该病毒进化的关键蛋白,且既不是分泌性蛋白也不是跨膜蛋白；有三个潜在的磷酸化位点,没有糖基化位点；.抗原表位主要集中在第2～12、第35-40、第49～53、第62-65、第72～75、第77～84位氨基酸,免疫原性良好。2. DuCV基因的克隆表达及纯化PCR特异性扩增ORF3基因后进行T-A克隆,再用EcoR Ⅰ和Xhol这两种内切酶消化后连接到表达载体上,成功构建了重组原核表达载体：pET-32a-ORF3和pGEX-6p-ORF3.重组载体分别转化进Rosetta (DE3) E.coli菌后用IPTG成功诱导表达了30 KD和35 KD的ORF3融合蛋白,与预期大小一致,对pET-32a-ORF3的最佳IPTG诱导浓度为0.8 mM,而对pGEX-6p-ORF3是0.2 mM,这两种融合蛋白都主要是以包涵体的形式存在。利用Ni2+亲和层析及包涵体洗涤和SDS-PAGE切胶回收相结合的方法分别纯化pET-32a-ORF3和pGEX-6p-ORF3重组蛋白,得到了高纯度的蛋白。Western Blot检测显示这两种蛋白均能够与鸭圆环病毒阳性血清特异性结合,表明它们都具有免疫学活性。3. DuCV ORF3单克隆抗体的制备纯化的pET-32a-ORF3蛋白经梯度复性后以100μg/只腹腔注射Balb/c雌性小鼠进行免疫,效价达到104后,将骨髓瘤细胞与其脾细胞按1：4的比例混匀,在PEG4000作用下进行融合反应。用建立的间接ELISA法(最佳抗原包被浓度1μg/孔)筛选阳性细胞,融合率为25%。经过3次有限稀释,最终达到100%的阳性,获得4株稳定分泌抗ORF3抗体的杂交瘤细胞(依次命名为1、2、3、4号)。利用ORF3阳性杂交瘤细胞制备腹水型抗体,抗体的效价在1：25600～1：102400之间,均为IgM亚类,并能够与ORF3蛋白特异性结合。4.间接竞争ELISA方法的建立与应用利用纯化的pET-32a-ORF3融合蛋白作为包被抗原,建立了稳定可靠的DuCV ORF3抗体的间接竞争ELISA免疫学检测方法。利用棋盘滴定法优化包被抗原和腹水单抗的最佳梯度,再确定出最优血清稀释度、竞争作用时间和cut-off判定值。结果表明该方法的最佳条件为pET-32a-ORF3纯化蛋白以1μg/包被,5%BSA封闭后,1：800的DuCV阳性血清37℃放置30 min,直接加入1：4000的腹水型单克隆抗体,混匀后37℃竞争反应40 min,HRP-羊抗鸭Ig G二抗孵育再TMB显色。该检测方法cut-off判定值为10.9%,只特异性的与DuCV阳性血清反应,灵敏度为血清稀释1：1600倍。将该方法与PCR检测方法对45份未知样品进行检测并比较,结果PCR检测出37个阳性,而本实验方法检测出25个阳性,且这25个样品在PCR检测中也均为阳性,符合率为100%。
[Abstract]:Duck circovirus is classified in the circoviridae circovirus genus, the virus ORF3 encoding protein function has not yet been explained. In order to prepare monoclonal antibody against this protein, to further explore the function of this protein to provide material, the DuCV ORF3 gene on the isolation and identification of the laboratory to carry out the following experiments: the bioinformatics analysis of DuCV ORF3 gene, ORF3 gene cloning, expression and purification of monoclonal antibody preparation, establishment and application of indirect competitive ELISA method, the results are as follows: analysis of GH01 strain was 1988 by DuCV 1. DuCV ORF3 biological information, ORF3 gene A, T, G, C contents were 21.89%, 25.25%, 24.58%, 28.28% 297bp, located in the whole genome, 399th to 103rd nucleotides, encoding 98 amino acids, random coil accounted for most of the molecular weight of about 11.6 KD. of the two protein secondary structure, content is about 62.3%, alpha helix and p- folding structure Accounted for 31.6% and 6.1%; no conserved signal peptide, transmembrane region, suggesting that the protein may be a key protein in the virus evolution, and neither secreted protein nor transmembrane protein; there are three potential phosphorylation sites, no glycosylation sites.; epitope mainly concentrated in second 12, article 35-40, article 62-65, forty-ninth ~ 53, seventy-second ~ 75, seventy-seventh ~ 84 amino acid, cloning and expression of immunogenicity.2. DuCV gene and purification of PCR specific ORF3 gene was amplified by T-A and cloned, EcoR I and Xhol two is connected to the expression vector after digestion, success to construct a Recombinant Prokaryotic expression vector pET-32a-ORF3 and recombinant vector pGEX-6p-ORF3. was transformed into Rosetta (DE3) E.coli strain by IPTG after successfully induced expression of 30 KD and 35 KD ORF3 fusion protein, consistent with the expected size, the best IPTG on pET-32a-ORF3 induced concentration For 0.8 mM, which is 0.2 mM for pGEX-6p-ORF3, the two kinds of fusion protein mainly existed in the form of inclusion body. By using the method of Ni2+ gel extraction combined with affinity chromatography and inclusion body washing and SDS-PAGE respectively. The pET-32a-ORF3 and pGEX-6p-ORF3 recombinant protein, the protein of high purity.Western Blot detection showed that the two proteins were capable of binding and duck circovirus positive serum specific, showed that they all have the immunological activity of purified.3. DuCV ORF3 monoclonal antibody against pET-32a-ORF3 protein was refolded by gradient with 100 g/ intraperitoneal injection of Balb/c female mice was up to 104 after the myeloma cells and spleen cells by 1:4 the mixing, the fusion reaction in the presence of PEG4000. With the developed ELISA method (the optimal antigen concentration of 1 g/ hole) screening positive cells, the fusion rate was 25%. after 3 times Limited dilution, eventually reaching 100% positive, obtained 4 hybridoma cell strains secreting anti ORF3 antibodies (designated as 1,2,3,4). The preparation of ascites antibody by ORF3 positive hybridoma cells, antibody titer was 1:25600 ~ 1:102400, were subtype IgM, and can be combined with ORF3 protein the establishment and application of.4. indirect competitive ELISA method using the purified pET-32a-ORF3 fusion protein as antigen to establish an indirect competitive DuCV method for immunological detection of ELISA antibody of ORF3 is stable and reliable. The chessboard titration optimization package was the best gradient antigen and monoclonal antibodies, and then determine the optimal serum dilution, competition time and cut-off to determine the value. The results showed that the best conditions of the method for purification of pET-32a-ORF3 protein in 1 g/ package, 5%BSA closed, 1:800 DuCV positive serum of 37 DEG C for 30 min, directly adding 1 Type 4000: ascites monoclonal antibody, mixed competition reaction 37 C 40 min, HRP- Ig G two anti Goat anti duck incubation TMB color. The detection method to determine cut-off value of 10.9%, a specificity and sensitivity of DuCV positive sera, sera were diluted 1:1600 times. The method with PCR the detection method for the detection of 45 samples and compared the results of 37 positive PCR was detected, and the experimental method to detect 25 positive, and the 25 samples in PCR assay were also positive, the coincidence rate was 100%.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【相似文献】