犬副流感病毒单克隆抗体的制备及单链抗体蛋白的表达和活性鉴定

发布时间：2018-04-16 18:27

本文选题：犬副流感病毒 + 单克隆抗体　；参考：《华中农业大学》2015年硕士论文

【摘要】：犬副流感病毒(Canine parainfluenza virus,CPIV)是引起犬传染性呼吸系统疾病(窝咳)的重要病原之一,目前几乎所有养犬国家均有此病流行。犬感染CPIV发病时往往伴随发烧、流涕、咳嗽等症状,容易与犬瘟热的症状混淆而延误治疗。因此,CPIV诊断、治疗试剂的研制非常重要。杂交瘤技术制备的单克隆抗体是目前最常用的疾病诊断治疗制剂,因此本研究使用浓缩的CPIV蛋白免疫小鼠,通过杂交瘤技术筛选单克隆抗体。相对杂交瘤技术制备的单抗而言,单链抗体蛋白具有相对分子量小、穿透力强等优点,在解析抗原抗体复合物结构、制备交叉保护作用抗体、检测病毒基因型变化等方面具有重要意义。因此,本研究建立了一套抗体基因扩增与表达的体系,保存了抗体基因;获得的抗体蛋白在一定程度上可作为天然抗体的替代品使用,为后续抗原抗体结构的解析、基因工程疫苗的研究奠定了基础。主要研究内容包括:1.单克隆抗体制备及生物学活性研究本研究使用PEG6000对Vero细胞扩增的病毒进行浓缩。TCID50试验测定浓缩前病毒毒价为2.6×105 PFU/ml,浓缩后病毒毒价为7×106 PFU/ml。血凝试验显示CPIV对1%猪红细胞具有血凝特性,血凝效价为23。将浓缩的病毒用弗氏佐剂乳化后免疫6周龄Babl/c小鼠。当免疫小鼠血清12800倍稀释后ELISA检测效价高于1.0时进行融合试验。三次亚克后共获得六株能够稳定分泌单克隆抗体的细胞株,分别命名为13E8、33B5、51D8、51E6、51G12和54D10。间接免疫荧光试验和Westernblot试验结果显示:13E8和51D8分泌针对P蛋白抗原的抗体,33B5、51E6、51G12和54D10分泌针对N蛋白抗原的抗体。为进一步分析针对N蛋白抗原的单克隆抗体的具体抗原域,分别构建N蛋白不同区域(1-509、40-509、204-50和40-375)的pc DNA3.1重组质粒,并于293T细胞中进行表达。间接免疫荧光试验结果显示,33B5和51G12的单抗识别抗原域位于N蛋白375-509位氨基酸内,而51E6和54D10的单抗识别抗原域位于N蛋白1-40位氨基酸内。2.单克隆抗体基因扩增、表达及单链抗体生物活性研究本研究成功获得了33B5、51D8和51E6 3株单克隆抗体基因,构建了p ET42b的重组质粒,并通过大肠杆菌进行原核表达。结果显示3种蛋白均表达在包涵体中。为获得有活性的目的抗体蛋白,本研究选取了33B5的抗体基因对其表达进行优化,包括:表达不同抗体区域、添加可溶性标签、更换表达载体和表达菌,调整表达时间、温度和诱导剂浓度。结果显示原核表达的33B5抗体蛋白均在包涵体中,且表达量很高。使用昆虫细胞表达33B5的抗体蛋白,结果显示抗体蛋白表达在昆虫细胞细胞质内或细胞膜上,且表达量很低。最终本试验通过尿素变性和梯度复性的办法获得了有活性的单链抗体蛋白33B5sc Fv。通过ELISA试验、CPIV感染Vero细胞后的间接免疫荧光试验以及浓缩CPIV的Westernblot试验分析后,我们证实了复性的33B5sc Fv蛋白具有跟天然抗体特异性一致的抗原结合能力。
[Abstract]:Canine parainfluenza virus (Canine parainfluenza virus) is one of the most important pathogens causing infectious respiratory diseases in dogs. At present, it is prevalent in almost all dog countries.CPIV infection in dogs is often accompanied by fever, runny, cough and other symptoms, easily confused with the symptoms of canine distemper and delayed treatment.Therefore, the development of CPIV diagnostic and therapeutic reagent is very important.The monoclonal antibody prepared by hybridoma technique is the most commonly used disease diagnosis and treatment preparation. So this study immunized mice with concentrated CPIV protein and screened monoclonal antibody by hybridoma technique.Compared with the monoclonal antibody prepared by hybridoma technique, the single-chain antibody protein has the advantages of relatively small molecular weight, strong penetration and so on. It is used to analyze the structure of antigen and antibody complex and to prepare cross-protection antibody.It is of great significance to detect the variation of virus genotypes.Therefore, a set of antibody gene amplification and expression system was established in this study, and the antibody gene was preserved. The obtained antibody protein can be used as a substitute for natural antibody to some extent, which can be used to analyze the structure of subsequent antigen and antibody.The research of genetic engineering vaccine has laid the foundation.The main research contents include: 1.Preparation and Biological activity of Monoclonal Antibodies; in this study, PEG6000 was used to concentrate the virus amplified from Vero cells. TCID50 assay was used to determine that the concentration of previrus was 2.6 脳 10 ~ 5 PFU / ml, and that of concentrated virus was 7 脳 10 ~ 6 PFU / ml.Hemagglutination test showed that CPIV had the characteristics of hemagglutination to 1% porcine erythrocytes, and the hemagglutination titer was 23. 5%.The concentrated virus was emulsified with Freund's adjuvant to immunize 6 week old Babl/c mice.The fusion test was carried out when the titer of ELISA was higher than 1.0 when the serum of immunized mice was 12800 times diluted.Six cell lines with stable secretion of monoclonal antibodies were obtained after three doses of subgram, and were named 13E833B5 51D81E6O51G12 and 54D10.The results of indirect immunofluorescence assay and Westernblot test showed that the antibody against P protein antigen was secreted by 51D8 and the antibody against P protein antigen ~ (33) B _ (51E _ (6)) ~ (51) G _ (12) and 54D10 secreted antibody against N protein antigen.In order to further analyze the specific antigen domain of monoclonal antibody against N protein antigen, the recombinant plasmids of PC DNA3.1 were constructed and expressed in 293T cells.The results of indirect immunofluorescence assay showed that the recognition antigen domain of mAbs of T33B5 and 51G12 was located in the amino acids of N-protein 375-509, while that of 51E6 and 54D10 was within the amino acids of N-protein 1-40.Amplification and expression of Monoclonal Antibody Gene and Biological activity of single chain Antibody. In this study, the monoclonal antibody genes of 33B5O51D8 and 51E6 3 strains were successfully obtained. The recombinant plasmid of p ET42b was constructed and expressed in E. coli.The results showed that all three proteins were expressed in inclusion bodies.In order to obtain the active target antibody protein, we selected the antibody gene of 33B5 to optimize its expression, including expressing different antibody regions, adding soluble label, changing expression vector and expressing bacteria, adjusting expression time.Temperature and inducer concentration.The results showed that the prokaryotic expression of 33B5 antibody protein was in the inclusion body and the expression level was very high.Using insect cells to express the antibody protein of 33B5, the results showed that the antibody protein was expressed in the cytoplasm of insect cell or on the cell membrane, and the amount of expression was very low.Finally, the reactive scFV protein 33B5sc FV was obtained by urea denaturation and gradient renaturation.The indirect immunofluorescence assay of Vero cells infected by ELISA assay and the analysis of Westernblot assay of CPIV concentration showed that the refolded 33B5sc Fv protein had antigen-binding ability consistent with natural antibody.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

【参考文献】