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相关词典网站：Keywordsporcine reproductive and respiratory syndrome virus, GP4 and GP5, multiepitopes string, mice, immune response
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&&&&&&&&猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome, PRRSV)引起的一种以母猪繁殖障碍和仔猪呼吸道疾病为主要特征的传染病。本研究采用重叠区扩增基因拼接法(gene splicing by overlap extension, SOEing)构建出两个含有PRRSV GP5和GP4优势抗原表位核苷酸片段的多表位串联基因,分析其真核表达重组质粒和原核表达产物在小鼠诱导的免疫反应。
采用SOEing PCR的方法将编码GP5的aa29～60和GP4的aa33～70核苷酸片段进行拼接,获得两个编码双表位多串联基因E54-2和E54-4。将E54-2和E54-4基因分别克隆至pcDNA3.1(+),构建出真核表达重组质粒pcDNA3-E54-2、pcDNA3-E54-4。采用SOEing PCR将E54-2基因与猪泛素基因(Ubiquiten)融合,获得融合基因Ub-E54-2,构建出真核重组表达质粒pcDNA3Ub-E54-2。将3种真核重组质粒分别转染COS7细胞,pcDNA3-E54-2和pcDNA3-E54-4分别转染P815细胞,间接免疫荧光试验表明,重组质粒在哺乳动物细胞中能瞬时和稳定表达目的蛋白E54-2和E54-4。
将E54-2和E54-4基因克隆至原核表达载体pGEX-6P-1,构建原核表达重组质粒pGEX-6P-E54-2和pGEX-6P-E54-4,经在大肠杆菌中诱导表达,蛋白表达量分别为40%和30%。采用包涵体洗涤和电洗脱纯化重组蛋白GST-E54-2和GST-E54-4。Western-Blotting分析结果表明,重组蛋白可与猪源PRRSV阳性血清和鼠源的抗GP4和GP5血清发生特异性反应。
用3种真核表达重组质粒分别免疫小鼠,结果表明,3种真核表达重组质粒均能诱导体液免疫反应,产生特异性的抗GP4、GP5和多表位串联肽抗体。在首免后第28天,pcDNA3-E54-2和pcDNA3-E54-4免疫组和对照组之间抗GP5抗体水平差异显著(p&0.05);首免后第49天,pcDNA3-E54-2和pcDNA3-E54-4免疫组之间抗GP5抗体差异显著(p&0.05);在首免后第28天,pcDNA3-E54-2和pcDNA3-E54-4免疫组和对照组之间抗GP4抗体水平差异显著(p&0.05)。4免后3周,pcDNA3-E54-2免疫组中和抗体效价为1:16,pcDNA3-E54-4免疫组中和抗体效价为1:28。
用纯化的融合蛋白GST-E54-2、GST-E54-4和表达GST-E54-2和GST-E54-4融合肽的基因工程全菌体分别免疫小鼠。经ELISA检测结果表明,在3免后1周,E54-2全菌免疫组和纯化蛋白免疫组抗GP4和GP5抗体效价分别为1:1,600和1:3,200;E54-4全菌免疫组和纯化蛋白免疫组抗GP4抗体效价为1:3,200,抗GP5抗体效价分别为1:12,800和1:6,400。中和抗体检测结果表明,在3免后1周,E54-2全菌免疫组和纯化蛋白免疫组抗体效价分别为1:22.2和1:19.9;E54-4全菌免疫组和E54-4纯化蛋白免疫组抗体效价为1:45.7和1:33.9,高于E54-2全菌免疫组和纯化蛋白免疫组。
综上结果表明,构建的PRRSV GP4、GP5多表位真核重组表达质粒在小鼠体内可诱导特异性抗体产生,原核表达的GP4、GP5多表位串联肽具有良好的免疫原性,有开发成疫苗的潜在价值。
&&&&Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease of swine caused by porcine reproductive and respiratory syndrome virus (PRRSV). This disease is characterized by reproductive failure in sows and respiratory illness in piglets. In the study, the gene fragments of two preponderant epitopes of GP5 and GP4 of PRRSV BJ-4 were amplified and fused by means of the gene splicing using overlap extension (gene SOEing) PCR technique in order to construct two multiepitopes fusion genes. The immune responses elicited by the eukaryotic recombinant plasmids expressing the two multiepitopes fusion genes and prokaryotic expressed multiepitopes peptides were analyzed in mice.The epitope areas of GP5 aa29~60 (named E5) and GP4 aa33~70 (named E4) were amplified and fused by gene SOEing PCR technique. Two new genes (E54-2 and E54-4) encoding the multiepitope peptides were generated. The two new genes were then cloned into the eukaryotic vector pcDNA3.1 to construct two eukaryotic recombinant plasmids, pcDNA3-E54-2 and pcDNA3-E54-4. The fusion gene Ub-E54-2, obtained by fusing the ubiquiten gene of porcine with the E54-2 gene using gene SOEing PCR, was inserted into pcDNA3.1 to construct eukaryotic recombinant plasmid pcDNA3Ub-E54-2. Indirect immunofluorescence assay was carried out following COS7 cells transfected by all three eukaryotic recombinant plasmids respectively and P815 cells transfected by the two former recombinant plasmids. The results indicated that the multiepitope peptides E54-2 and E54-4 could be instantaneously and steadily expressed in mammalian cells.The E54-2 and E54-4 genes were cloned into the prokaryotic expression vector pGEX-6P-l respectively to construct two prokaryotic recombinant expression plasmids, pGEX-6P-E54-2 and pGEX-6P-E54-4. The two prokaryotic recombinant expression plasmids were expressed in Escherichia coli BL21 (DE3). The recombinant fusion proteins GST-E54-2 and GST-E54-4 (expressed by pGEX-6P-E54-2 and pGEX-6P-E54-4, respectively) could amount to 40% and 30% of the total mass of bacterial proteins. The recombinant proteins GST-E54-2 and GST-E54-4 were purified through denaturation and refolding of inclusion bodies and electro-elution assay. Western-Blotting analysis indicated that the purified proteins GST-E54-2 and GST-E54-4 could be recognized by the anti-PRRSV serum of porcine, anti-GP4 and anti-GP5 serum of mice.The mice were immunized with the three eukaryotic recombinant plasmids, respectively. It was shown that the three plasmids were able to induce the humoral immune responses. The specific anti-GP4, anti-GP5 and anti-E54-2 peptide antibodies could be deteced. At day 28 post primary immunization (PPI), the antibodies against GP5 in groups immunized with pcDNA3-E54-2 and pcDNA3-E54-4 were significantly higher than that of the control group (p&0.05). There was a significant difference in response levels of antibodies against GP5 between pcDNA3-E54-2 group and pcDNA3-E54-4 group at day 49 PPI (p&0.05). At day 28 PPI, the antibodies against GP4 showed significant differences between the control group and pcDNA3-E54-2 or pcDNA3-E54-4 groups (p&0.05). After three weeks following the fourth immunization, the titre of viral neutralizing (VN)antibodies in groups immunized with pcDNA3-E54-2 or pcDNA3-E54-4 were 1:16, 1:28, respectively.Mice were immunized with the purified fusion proteins (GST-E54-2 and GST-E54-4), and genetically engineered bacteria expressing the fusion proteins GST-E54-2 and GST-E54-4, respectively. The results of ELISA analysis exhibited that the anti-GP4 and anti-GP5 antibody titres in the E54-2 bacteria group and purified protein group were 1:1,600 and 1:3,200 respectively, anti-GP4 antibody titres of the E54-4 bacteria and purified protein group were 1:3,200, anti-GP5 antibody titres of the E54-4 bacteria and purified protein group were 1:12,800 and 1:6,400 respectively one week after the third immunization. The data of VN antibodies detection demonstrated that the titre of VN antibodies of the E54-2 bacteria group was 1:22.2, and that of puri
&&&&&&&&PRRSV GP4和GP5多表位串联基因的表达及在小鼠诱导的免疫反应摘要4-5ABSTRACT5插图与附表清单9-11缩略词表11-12第一章 引言12-16&&&&1.1 国内外研究概况12-14&&&&1.2 研究目的与意义14-15&&&&1.3 研究内容与技术路线15&&&&1.4 研究目标15-16第二章 PRRSV GP5和GP4多表位基因串联及真核重组质粒的构建16-41&&&&2.1 引言16&&&&2.2 材料与方法16-29&&&&2.3 结果与分析29-38&&&&2.4 讨论38-39&&&&2.5 小结39-41第三章 PRRSV GP4和GP5多表位串联肽的原核表达与纯化41-57&&&&3.1 引言41&&&&3.2 材料与方法41-48&&&&3.3 结果与分析48-54&&&&3.4 讨论54-56&&&&3.5 小结56-57第四章 表达PRRSV GP4和GP5多表位串联肽真核重组质粒在小鼠诱导的免疫反应57-75&&&&4.1 引言57-58&&&&4.2 材料与方法58-66&&&&4.3 结果与分析66-72&&&&4.4 讨论72-74&&&&4.5 小结74-75第五章 原核表达PRRSV GP4和GP5多表位串联肽在小鼠体内的免疫反应75-88&&&&5.1 引言75-76&&&&5.2 材料与方法76-80&&&&5.3 结果与分析80-85&&&&5.4 讨论85-87&&&&5.5 小结87-88第六章 结论88-89第七章 创新点89-90第八章 文献综述90-100&&&&8.1 表位的研究状况90-92&&&&8.2 表位疫苗的研究92-96&&&&8.3 提高表位疫苗的免疫效果措施96-100参考文献100-111致谢111-112作者简介112
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the site on the surface of an antigen molecule to which an antibody attaches itself
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【近义词】
抗原决定簇
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