本文選題：奶牛　切入點：炎性疾病　出處：《黑龍江八一農(nóng)墾大學》2015年碩士論文　論文類型：學位論文

【摘要】：在奶牛疾病中，亞臨床炎性疾病因臨床癥狀不明顯，早期不易被發(fā)現(xiàn)，給奶牛養(yǎng)殖業(yè)帶來嚴重的經(jīng)濟損失，已經(jīng)成為制約奶牛養(yǎng)殖業(yè)健康發(fā)展的一個瓶頸問題。因此，奶牛亞臨床炎性疾病早期預警方法的研究具有重要科學意義及應用價值。牛結合珠蛋白（bovinehaptoglobin，BoHp）是一種血漿急相蛋白，是多種奶牛感染性疾病的生物標記物，在炎性疾病早期診斷中有潛在的應用價值。 在本研究中，以純化的pirBoHp重組蛋白作為抗原，免疫BALB/c小鼠，利用淋巴細胞雜交瘤技術，制備了5株BoHp單克隆抗體，分別命名為1B3、6D6、6D3、6B7和1C1。Westernblot結果顯示，在制備的5株BoHp單克隆抗體中，1B3、6D6、6D3和6B7能識別牛血漿中天然BoHp的α鏈。進一步抗體亞型鑒定結果表明，1B3、6D6、6D3和6B7抗體亞型均為IgG1，輕鏈型為kappa鏈。利用親和層析技術對BoHp單克隆抗體1B3、6D6、6D3和6B7腹水中的IgG蛋白進行了純化，進一步利用改良過碘酸鈉標記法，對純化的4株BoHp單克隆抗體進行辣根過氧化物酶（HRP）標記，，利用純化的BoHp單克隆抗體和HRP標記抗體，建立BoHp的定量ELISA檢測方法。敏感性試驗結果顯示，BoHp定量ELISA最低檢測濃度為31.25ng/mL。臨床樣品檢測結果顯示，本研究建立的pirBoHp定量夾心ELISA方法與商品化ELISA符合率為75%。利用檸檬酸三鈉還原法，制備膠體金顆粒，并對BoHp單克隆抗體進行金標記，進一步利用純化的BoHp單克隆抗體和羊抗鼠IgG印跡硝酸纖維素膜分別作為檢測線和質(zhì)控線，組裝成檢測BoHp的膠體金試紙條。條件優(yōu)化試驗結果顯示，BoHp單克隆抗體1B3作為金標抗體，6D3作為檢測抗體時，膠體金試紙條的檢測效果最佳。敏感性試驗結果顯示，組裝的BoHp膠體金試紙條最低檢測濃度為0.45μg/mL。臨床試驗結果顯示，48份不同跛行程度奶牛血清樣品中43份樣品出現(xiàn)信號，其中14份樣品出現(xiàn)強信號，其陽性率為89.58%。表明膠體金試紙條能特異性識別牛血清中BoHp。 本研究擬通過已篩選的奶牛亞臨床炎性疾病預警Marker-牛結合珠蛋白（BoHp）作為研究靶標，制備BoHp蛋白的單克隆抗體，建立奶牛炎性疾病預警Marker BoHp的診斷方法，通過臨床效果評價，明確BoHp作為奶牛亞臨床炎性疾病預警Marker的可行性，為奶牛亞臨床炎性疾病的早期預警及治療提供有效工具。
[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 偽 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.
【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23

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