本文選題：犬副流感病毒 + 單克隆抗體��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：犬副流感病毒(Canine parainfluenza virus,CPIV)是引起犬傳染性呼吸系統(tǒng)疾病(窩咳)的重要病原之一,目前幾乎所有養(yǎng)犬國家均有此病流行。犬感染CPIV發(fā)病時往往伴隨發(fā)燒、流涕、咳嗽等癥狀,容易與犬瘟熱的癥狀混淆而延誤治療。因此,CPIV診斷、治療試劑的研制非常重要。雜交瘤技術(shù)制備的單克隆抗體是目前最常用的疾病診斷治療制劑,因此本研究使用濃縮的CPIV蛋白免疫小鼠,通過雜交瘤技術(shù)篩選單克隆抗體。相對雜交瘤技術(shù)制備的單抗而言,單鏈抗體蛋白具有相對分子量小、穿透力強等優(yōu)點,在解析抗原抗體復(fù)合物結(jié)構(gòu)、制備交叉保護(hù)作用抗體、檢測病毒基因型變化等方面具有重要意義。因此,本研究建立了一套抗體基因擴增與表達(dá)的體系,保存了抗體基因;獲得的抗體蛋白在一定程度上可作為天然抗體的替代品使用,為后續(xù)抗原抗體結(jié)構(gòu)的解析、基因工程疫苗的研究奠定了基礎(chǔ)。主要研究內(nèi)容包括:1.單克隆抗體制備及生物學(xué)活性研究本研究使用PEG6000對Vero細(xì)胞擴增的病毒進(jìn)行濃縮。TCID50試驗測定濃縮前病毒毒價為2.6×105 PFU/ml,濃縮后病毒毒價為7×106 PFU/ml。血凝試驗顯示CPIV對1%豬紅細(xì)胞具有血凝特性,血凝效價為23。將濃縮的病毒用弗氏佐劑乳化后免疫6周齡Babl/c小鼠。當(dāng)免疫小鼠血清12800倍稀釋后ELISA檢測效價高于1.0時進(jìn)行融合試驗。三次亞克后共獲得六株能夠穩(wěn)定分泌單克隆抗體的細(xì)胞株,分別命名為13E8、33B5、51D8、51E6、51G12和54D10。間接免疫熒光試驗和Westernblot試驗結(jié)果顯示:13E8和51D8分泌針對P蛋白抗原的抗體,33B5、51E6、51G12和54D10分泌針對N蛋白抗原的抗體。為進(jìn)一步分析針對N蛋白抗原的單克隆抗體的具體抗原域,分別構(gòu)建N蛋白不同區(qū)域(1-509、40-509、204-50和40-375)的pc DNA3.1重組質(zhì)粒,并于293T細(xì)胞中進(jìn)行表達(dá)。間接免疫熒光試驗結(jié)果顯示,33B5和51G12的單抗識別抗原域位于N蛋白375-509位氨基酸內(nèi),而51E6和54D10的單抗識別抗原域位于N蛋白1-40位氨基酸內(nèi)。2.單克隆抗體基因擴增、表達(dá)及單鏈抗體生物活性研究本研究成功獲得了33B5、51D8和51E6 3株單克隆抗體基因,構(gòu)建了p ET42b的重組質(zhì)粒,并通過大腸桿菌進(jìn)行原核表達(dá)。結(jié)果顯示3種蛋白均表達(dá)在包涵體中。為獲得有活性的目的抗體蛋白,本研究選取了33B5的抗體基因?qū)ζ浔磉_(dá)進(jìn)行優(yōu)化,包括:表達(dá)不同抗體區(qū)域、添加可溶性標(biāo)簽、更換表達(dá)載體和表達(dá)菌,調(diào)整表達(dá)時間、溫度和誘導(dǎo)劑濃度。結(jié)果顯示原核表達(dá)的33B5抗體蛋白均在包涵體中,且表達(dá)量很高。使用昆蟲細(xì)胞表達(dá)33B5的抗體蛋白,結(jié)果顯示抗體蛋白表達(dá)在昆蟲細(xì)胞細(xì)胞質(zhì)內(nèi)或細(xì)胞膜上,且表達(dá)量很低。最終本試驗通過尿素變性和梯度復(fù)性的辦法獲得了有活性的單鏈抗體蛋白33B5sc Fv。通過ELISA試驗、CPIV感染Vero細(xì)胞后的間接免疫熒光試驗以及濃縮CPIV的Westernblot試驗分析后,我們證實了復(fù)性的33B5sc Fv蛋白具有跟天然抗體特異性一致的抗原結(jié)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3

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