本文關(guān)鍵詞：禽源H1N1亞型豬流感病毒HA蛋白特異性單克隆抗體的制備　出處：《山東農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

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【摘要】：豬流感(Swine Influenza,SI)是由正粘病毒科A型流感病毒屬的豬流感病毒(Swine Influenza virus,SIV)引起的一種豬的高度接觸的急性傳染性呼吸道疾病。目前為止已發(fā)現(xiàn)的SIV有H1N1、H1N2、H1N7、H2N3、H3N1、H3N2、H3N6、H4N6、H5N1及H9N2等多種亞型,禽源H1N1亞型豬流感病毒在我國豬群中流行的各亞型豬流感病毒中占有重要地位。本研究首先以禽源H1N1亞型豬流感病毒A/swine/Shanghai/1/2014(H1N1)(簡稱SH1)為試驗材料,接種MDCK細(xì)胞,培養(yǎng)72h內(nèi),待出現(xiàn)細(xì)胞病變后收集細(xì)胞上清。用蔗糖梯度離心的方法,純化病毒。進(jìn)行間接ELISA方法的建立,用于針對禽源H1N1亞型豬流感病毒抗體的檢測及篩選陽性雜交瘤細(xì)胞。研究表明,抗原包被濃度為5.725μg/ml,4℃過夜;陰、陽性血清作1:200稀釋;樣品最佳反應(yīng)作用條件為37℃,1h;酶標(biāo)二抗最佳反應(yīng)作用條件也為37℃,1h。該方法與H3N2亞型豬流感病毒、豬繁殖與呼吸綜合征病毒、豬瘟病毒、偽狂犬病毒等重要豬病原的陽性血清不發(fā)生交叉反應(yīng)。因此,本研究建立的禽源H1N1亞型豬流感病毒間接ELISA抗體檢測方法具有特異性好、操作簡便、快捷、結(jié)果準(zhǔn)確等特點。用4%甲醛對純化后的病毒進(jìn)行滅活,與弗氏佐劑混合乳化后皮下多點注射,免疫6-8周齡BALB/c雌性小鼠,每只小鼠100μg。之后每隔兩個周免疫一次,免疫劑量相同。二次免疫后,眼球采血,用ELISA方法測其血清抗體效價。選擇抗體效價高的小鼠,加強(qiáng)免疫后取小鼠脾細(xì)胞與SP2/0細(xì)胞進(jìn)行融合,制備雜交瘤細(xì)胞。用HI和ELISA方法對雜交瘤細(xì)胞上清進(jìn)行檢測,篩選出陽性細(xì)胞孔,再用有限稀釋法對陽性細(xì)胞株進(jìn)行亞克隆培養(yǎng),最后獲得2株H1亞型特異性的雜交瘤細(xì)胞株:1D9、3C7。經(jīng)鑒定兩株單克隆抗體均能與H1N1亞型豬流感病毒發(fā)生特異性反應(yīng),且與其他亞型豬流感病毒和其他豬病病原不反應(yīng),其中3C7為針對HA蛋白的特異性單抗,為下一步建立H1N1亞型豬流感的診斷方法提供了物質(zhì)保障。
[Abstract]:Swine influenza virus (Swine Influenzasiae) is a swine influenza virus belonging to the genus Orthomyxoviridae, Swine Influenza virus. SIV has been found to be a highly exposed acute respiratory disease in pigs. So far, the SIV has been found to have H2N7H2N3H3N1H3N2. H3N6, H4N6, H5N1, H9N2 and other subtypes. Avian H1N1 subtype swine influenza virus plays an important role in the swine influenza viruses of various subtypes in Chinese pigs. In this study, the avian H1N1 subtype swine influenza virus A / R / Shang was first studied. Hali / R / 2014. H1N _ 1 (abbreviated as SH _ 1) is a kind of experimental material. MDCK cells were inoculated and cultured within 72 hours. Cell supernatants were collected after cytopathic changes. The virus was purified by sucrose gradient centrifugation. Indirect ELISA method was established. It was used to detect and screen positive hybridoma cells against avian H1N1 subtype swine influenza virus antibody. The concentration of antigen coating was 5.725 渭 g / ml ~ 4 鈩，

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