【作者】 张鹏；

【导师】 马卫明；

【作者基本信息】 山东农业大学 ， 兽医（专业学位）， 2016， 硕士

【摘要】 猪流感(Swine Influenza,SI)是由正粘病毒科A型流感病毒属的猪流感病毒(Swine Influenza virus,SIV)引起的一种猪的高度接触的急性传染性呼吸道疾病。目前为止已发现的SIV有H1N1、H1N2、H1N7、H2N3、H3N1、H3N2、H3N6、H4N6、H5N1及H9N2等多种亚型,禽源H1N1亚型猪流感病毒在我国猪群中流行的各亚型猪流感病毒中占有重要地位。本研究首先以禽源H1N1亚型猪流感病毒A/swine/Shanghai/1/2014(H1N1)(简称SH1)为试验材料,接种MDCK细胞,培养72h内,待出现细胞病变后收集细胞上清。用蔗糖梯度离心的方法,纯化病毒。进行间接ELISA方法的建立,用于针对禽源H1N1亚型猪流感病毒抗体的检测及筛选阳性杂交瘤细胞。研究表明,抗原包被浓度为5.725μg/ml,4℃过夜;阴、阳性血清作1:200稀释;样品最佳反应作用条件为37℃,1h;酶标二抗最佳反应作用条件也为37℃,1h。该方法与H3N2亚型猪流感病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、伪狂犬病毒等重要猪病原的阳性血清不发生交叉反应。因此,本研究建立的禽源H1N1亚型猪流感病毒间接ELISA抗体检测方法具有特异性好、操作简便、快捷、结果准确等特点。用4%甲醛对纯化后的病毒进行灭活,与弗氏佐剂混合乳化后皮下多点注射,免疫6-8周龄BALB/c雌性小鼠,每只小鼠100μg。之后每隔两个周免疫一次,免疫剂量相同。二次免疫后,眼球采血,用ELISA方法测其血清抗体效价。选择抗体效价高的小鼠,加强免疫后取小鼠脾细胞与SP2/0细胞进行融合,制备杂交瘤细胞。用HI和ELISA方法对杂交瘤细胞上清进行检测,筛选出阳性细胞孔,再用有限稀释法对阳性细胞株进行亚克隆培养,最后获得2株H1亚型特异性的杂交瘤细胞株:1D9、3C7。经鉴定两株单克隆抗体均能与H1N1亚型猪流感病毒发生特异性反应,且与其他亚型猪流感病毒和其他猪病病原不反应,其中3C7为针对HA蛋白的特异性单抗,为下一步建立H1N1亚型猪流感的诊断方法提供了物质保障。更多还原

【Abstract】 Swine Influenza(SI) is a highly exposed acute infectious respiratory disease of swine,caused by Swine Influenza Virus(SIV) which is orthomyxoviridae type A influenza virus. At present there has been many subtypes of SIV, consisting of H1N1 、 H1N2 、 H1N7 、H2N3、H3N1、H3N2、H3N6、H4N6、H5N1 and H9N2. The Avian-like H1N1 subtype SIV has played an important role in the prevalence of SI of all kinds of subtypes in China.In the research, cultivating the MDCK cells for 72 hours which is inoculated by H1N1 subtype SIV and collect the supernatant after cytopathic effect. The SIV was purified by sucrose gradient centrifugation. To establish indirect ELISA method,for detection of antibodies of Avian-like H1N1 subtype SIV and screening positive hybridoma. The concentration of coated antigen is 5.725μg/ml, 4℃ for night. Negative and positive serums were diluted as 1:200; The best reaction condition of samples and enzyme labeled second antibody are 37℃for 1h. The cross-reaction of established method with positive serums of H3N2 SIV, PRRSV, HCV and PRV is bad. In conclusion this method established by indirected ELISA is specific simple, rapid and accurate.Inactivate purified virus with 4% formaldehyde and 6-8-week BALB/C female mice are injected at multiple sites subcutaneous at 100μg per mouse after mixed with freund’s adjuvant emulsion. Secondary Immunization with the same dose after two weeks. After secondary immunization collecting the blood from mice and measure the serum antibody titers with ELISA method. Strengthening immunization with mice of high antibody titers; collecting spleen cells which must be mixed with SP2/0 cells to make the hybridoma cells. Checking titers of the supernatant of hybridoma cells and screening positive cells holes. Cloning and cultivating positive cells with a limited dilution method to make 2 special hybridoma cells of H1 SIV, as 1D9,3C7. The result was showed two strains of monoclonal antibodies can react specificity with H1N1 SIV but with other subtypes of SIV or porcine pathogen. What’s more,3C7 is a special sheet resistance against HA protein and which can provide material insurance for establishing the detection method of H1N1 subtype SI.更多还原