牛结合珠蛋白单克隆抗体的制备及初步应用

发布时间：2018-03-06 23:20

  本文选题：奶牛　切入点：炎性疾病　出处：《黑龙江八一农垦大学》2015年硕士论文　论文类型：学位论文

【摘要】：在奶牛疾病中，亚临床炎性疾病因临床症状不明显，早期不易被发现，给奶牛养殖业带来严重的经济损失，已经成为制约奶牛养殖业健康发展的一个瓶颈问题。因此，奶牛亚临床炎性疾病早期预警方法的研究具有重要科学意义及应用价值。牛结合珠蛋白（bovinehaptoglobin，BoHp）是一种血浆急相蛋白，是多种奶牛感染性疾病的生物标记物，在炎性疾病早期诊断中有潜在的应用价值。 在本研究中，以纯化的pirBoHp重组蛋白作为抗原，免疫BALB/c小鼠，利用淋巴细胞杂交瘤技术，制备了5株BoHp单克隆抗体，分别命名为1B3、6D6、6D3、6B7和1C1。Westernblot结果显示，在制备的5株BoHp单克隆抗体中，1B3、6D6、6D3和6B7能识别牛血浆中天然BoHp的α链。进一步抗体亚型鉴定结果表明，1B3、6D6、6D3和6B7抗体亚型均为IgG1，轻链型为kappa链。利用亲和层析技术对BoHp单克隆抗体1B3、6D6、6D3和6B7腹水中的IgG蛋白进行了纯化，进一步利用改良过碘酸钠标记法，对纯化的4株BoHp单克隆抗体进行辣根过氧化物酶（HRP）标记，，利用纯化的BoHp单克隆抗体和HRP标记抗体，建立BoHp的定量ELISA检测方法。敏感性试验结果显示，BoHp定量ELISA最低检测浓度为31.25ng/mL。临床样品检测结果显示，本研究建立的pirBoHp定量夹心ELISA方法与商品化ELISA符合率为75%。利用柠檬酸三钠还原法，制备胶体金颗粒，并对BoHp单克隆抗体进行金标记，进一步利用纯化的BoHp单克隆抗体和羊抗鼠IgG印迹硝酸纤维素膜分别作为检测线和质控线，组装成检测BoHp的胶体金试纸条。条件优化试验结果显示，BoHp单克隆抗体1B3作为金标抗体，6D3作为检测抗体时，胶体金试纸条的检测效果最佳。敏感性试验结果显示，组装的BoHp胶体金试纸条最低检测浓度为0.45μg/mL。临床试验结果显示，48份不同跛行程度奶牛血清样品中43份样品出现信号，其中14份样品出现强信号，其阳性率为89.58%。表明胶体金试纸条能特异性识别牛血清中BoHp。 本研究拟通过已筛选的奶牛亚临床炎性疾病预警Marker-牛结合珠蛋白（BoHp）作为研究靶标，制备BoHp蛋白的单克隆抗体，建立奶牛炎性疾病预警Marker BoHp的诊断方法，通过临床效果评价，明确BoHp作为奶牛亚临床炎性疾病预警Marker的可行性，为奶牛亚临床炎性疾病的早期预警及治疗提供有效工具。
[Abstract]:In dairy cow disease, the subclinical inflammatory disease is not obvious because of the clinical symptoms, it is not easy to be discovered in the early stage, which brings serious economic loss to the dairy cattle breeding industry, and has become a bottleneck problem that restricts the healthy development of the dairy cattle breeding industry. The study of early warning method for subclinical inflammatory diseases in dairy cattle is of great scientific significance and application value. Bovine haptoglobin (BoHpP) is a kind of plasma rapid-phase protein and a biomarker of various infectious diseases in dairy cattle. It has potential application value in early diagnosis of inflammatory diseases. In this study, the purified pirBoHp recombinant protein was used as antigen to immunize BALB/c mice. Five monoclonal antibodies to BoHp were prepared by lymphocyte hybridoma technique. The monoclonal antibodies were named as 1B3O6D6D6D3O6B7 and 1C1. The 伪 chain of natural BoHp in bovine plasma can be identified by 1B3O6D6D6D3 and 6B7 among the five BoHp monoclonal antibodies prepared. The results of further antibody subtype identification showed that the antibody subtypes were IgG1, and the light chain was kappa chain. Affinity chromatography was used to detect BoHp by affinity chromatography. The IgG protein was purified from the ascites of 1B3, 6D6, 6D3 and 6B7. The purified monoclonal antibodies of BoHp were labeled by horseradish peroxidase (HRP), and the purified monoclonal antibodies of BoHp and HRP were labeled by modified sodium periodate labeling method. A quantitative ELISA detection method for BoHp was established. The sensitivity test results showed that the lowest detection concentration of ELISA was 31.25 ng / mL. The coincidence rate between pirBoHp quantitative sandwich ELISA method and commercial ELISA was 75%. Colloidal gold particles were prepared by tri-sodium citrate reduction method and labeled with BoHp monoclonal antibody. Further more, purified BoHp monoclonal antibody and sheep anti-mouse IgG imprinted nitrocellulose membrane were used as detection line and quality control line, respectively. A colloidal gold test strip was assembled for the detection of BoHp. The results of condition optimization test showed that when the monoclonal antibody 1B3 was used as the gold-labeled antibody 6D3 as the detection antibody, the detection effect of the colloidal gold strip was the best. The sensitivity test showed that, The minimum detectable concentration of BoHp colloidal gold test strip was 0.45 渭 g / mL. The clinical results showed that 43 of 48 serum samples with different lameness degree showed signal, 14 of which showed strong signal. The positive rate was 89.58. The results showed that the colloidal gold strip could specifically identify BoHpin in bovine serum. The aim of this study was to prepare monoclonal antibodies against BoHp protein and to establish a diagnostic method for early warning of Marker BoHp of dairy cow inflammatory disease by using the screened subclinical inflammatory disease warning marker (Marker-BoHp) as the research target, and to evaluate the clinical effect. To determine the feasibility of BoHp as an early warning Marker for subclinical inflammatory diseases in dairy cattle, and to provide an effective tool for early warning and treatment of subclinical inflammatory diseases in dairy cattle.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

【参考文献】

相关期刊论文 前2条

1 许腊梅;冯万宇;王岩;王志明;段莹莹;侯振中;;奶牛子宫内膜炎研究进展[J];动物医学进展;2008年08期

2 郭东华;祝东彬;孙东波;郭婷;赵秀宇;冉旭华;武瑞;;奶牛腐蹄病诊断方法研究进展[J];中国畜牧兽医;2011年05期

相关博士学位论文 前2条

1 彭伏虎;禽流感与新城疫胶体金免疫层析快速检测技术及初步应用研究[D];华中农业大学;2008年

2 李德军;奶牛隐性和临床型子宫内膜炎病理机制研究[D];华中农业大学;2010年

本文编号：1576944