發(fā)布時間：2019-04-24 02:05

【摘要】：病毒感染所引起的呼吸道疾病已經(jīng)成為世界性的常見疾病之一。人博卡病毒(HBoV)作為引起呼吸道感染的常見病毒之一,雖然發(fā)現(xiàn)相對較晚,但受到科研工作者的廣泛關(guān)注。而有效的診斷和治療需要具有高效價的抗體,才能建立系統(tǒng)的方法,單克隆抗體是病原檢測和診斷的主要工具。目前,有關(guān)HBoV單克隆抗體制備的研究很少,因此,HBoV單克隆抗體的制備對于HBoV的診斷和所引起的疾病的治療有重要意義。 本研究利用強大的原核表達載體pET-30a構(gòu)建重組質(zhì)粒,通過大腸桿菌表達系統(tǒng)表達重組VP2蛋白,通過柱上變復(fù)性純化目的蛋白。利用血清檢查抗原。以純化蛋白作為抗原,免疫小鼠,利用雜交瘤技術(shù)及間接ELISA方法篩選陽性雜交瘤細胞,小鼠體內(nèi)誘導(dǎo)產(chǎn)生單克隆抗體,并對單克隆抗體進行鑒定。 結(jié)果表明,構(gòu)建出HBoVVP2基因的重組質(zhì)粒,表達并純化得到了重組HBoVVP2蛋白,利用呼吸道感染患者血清檢測抗原,陽性率為77.7%,與國內(nèi)外檢出率基本一致,說明抗原較好。利用雜交瘤技術(shù)篩選得到了穩(wěn)定細胞株,該細胞株可以分泌單克隆抗體。利用該株細胞在小鼠腹部誘導(dǎo)產(chǎn)生具有單克隆抗體的腹水,根據(jù)檢測和鑒定,所產(chǎn)生的單克隆抗體為IgG抗體,具有較好抗原抗體結(jié)合能力,效價達到1：4×105。 本實驗得到的單克隆抗體,有益于對HBoV的深入研究。該研究結(jié)果對HBoV診斷、治療和進一步研究奠定基礎(chǔ)。
[Abstract]:Respiratory diseases caused by viral infection have become one of the most common diseases in the world. Human Boca virus (HBoV), one of the common viruses causing respiratory tract infection, has been paid more and more attention by researchers although it was found relatively late. Effective diagnosis and treatment requires high titer antibodies to establish a systematic method. Monoclonal antibodies are the main tools for the detection and diagnosis of pathogens. At present, there are few studies on the preparation of monoclonal antibodies against HBoV. Therefore, the preparation of monoclonal antibodies against HBoV is of great significance for the diagnosis of HBoV and the treatment of diseases caused by it. In this study, a powerful prokaryotic expression vector pET-30a was used to construct the recombinant plasmid, and the recombinant VP2 protein was expressed by E. coli expression system. The target protein was purified by renaturation on the column. Use the serum to test the antigen. The purified protein was used as antigen to immunize mice. The positive hybridoma cells were screened by hybridoma technique and indirect ELISA method. The monoclonal antibodies were induced to produce monoclonal antibodies in mice and the monoclonal antibodies were identified. The results showed that the recombinant plasmid of HBoVVP2 gene was constructed, and the recombinant HBoVVP2 protein was expressed and purified. The positive rate of the recombinant HBoVVP2 protein was 77. 7% by using the sera of patients with respiratory tract infection, which was basically consistent with the detection rate at home and abroad, indicating that the antigen was better. Stable cell lines were obtained by hybridoma technique, which could secrete monoclonal antibodies. The cell strain was used to induce ascites with monoclonal antibody in the abdomen of mice. According to the detection and identification, the monoclonal antibody produced was IgG antibody, which had good antigen-antibody binding ability, and the titer was up to 1:4 脳 10 ~ 5. The monoclonal antibodies obtained in this experiment are beneficial to the in-depth study of HBoV. The results of this study lay the foundation for the diagnosis, treatment and further study of HBoV.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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