【作者】 段张秀；

【导师】 宁长申； 张龙现；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 为建立弓形虫病标准、准确、快速的免疫诊断体系及亚单位疫苗的研制,为弓形虫抗原的分离和纯化及探索弓形虫的保护性免疫等研究提供适宜的工具,本研究利用经淋巴细胞分离液密度梯度离心、微孔滤膜过滤、多次低速离心提纯的弓形虫速殖子,再通过反复冻融和超声波处理后作为抗原免疫Balb/c小鼠6次,获得针对弓形虫速殖子的抗体。间接ELISA检测结果显示,6免后抗体效价均达到1：6400以上。本次建立的间接ELISA方法,其主要条件如下：0.5μg·mL-1的弓形虫速殖子为包被抗原,1%牛血清白蛋白为封闭剂,1：5000稀释HRP标记羊抗鼠IgG作为二抗,TMB(3,3,5,5-四甲基联苯)作为底物。实验证明,该方法特异性强,有较好的重复性。用该法对弓形虫速殖子免疫小鼠的血清抗体及其杂交瘤细胞单抗上清进行检测,获得满意的效果。以PEG2000为促融剂,取效价高的免疫小鼠脾细胞与NSO骨髓瘤细胞融合。经间接ELISA方法筛选阳性孔,有限稀释法进行3-4次克隆和亚克隆,获得4株能稳定分泌抗弓形虫单克隆抗体的杂交瘤细胞株(3A3、3G3、4F4、4G3),1株单抗腹水(3A3)。经鉴定,4株杂交瘤细胞株的上清效价分别在1：800-12800之间,1株腹水(3A3)效价在1：51200,且稳定性良好,单抗亚类为IgG1,与猪等孢子球虫、猪隐孢子虫、猪旋毛虫、猪蛔虫等无特异性。采用免疫荧光法对腹水进行初步鉴定,发现均为抗速殖子单抗。单抗的腹水提纯后进行SDS-PAGE和Westen-blotting分析,单抗的分子量均约为45kDa。用3A3A5单抗包板酶标板与猪抗弓形虫抗体建立夹心ELISA方法,单抗的最佳稀释度为1：800,猪抗弓形虫高免血清的最佳稀释度为1：100-200。特异性试验表明：该方法对于同属于孢子虫属的猪等孢子球虫、猪隐孢子虫、猪旋毛虫、猪蛔虫有较高的识别力,其OD450nm值则显著偏低。该方法不受其它外界因素的影响,灵敏度高,特异性强,对于临床诊断和公共卫生学将是一种简便、可靠的检测方法,为下一步建立弓形虫病快速诊断体系奠定了基础。更多还原

【Abstract】 The aim of my research is to lay foundations for diagnosis and preparation for subunit vaccine of toxoplasmosis,and Separation and purification of antigens and Explore of Protective immunity.The tachyzoites of Toxoplasma gondii RH strain were purified by discontinuous Fluid lymphocytes gradients,then which was treated by filtration and centrifugation,freeze-thawed and sonicated.Balb/c mice were immunized for six times with purified Tachyzoites of Toxoplasma gondii and the ELISA titers were all above 1:6400 in the end.Indirect Enzyme-linked Immunosorbent Assayz(ELISA) method was developed to detect antibodies against Toxoplasma gondii.The optimum conditions of the ELISA are set as follows:Toxoplasma gondii crude antigen diluted by carbonate buffer solution(CBS,pH 9.6,0.05mol/L)in 1:20 as a coated antigen dissolved,1% BSA as a blocking agent post-coating,horse radish heroxidase-labeled sheep anti-mouse IgG(HRP-anti IgG)diluted by PBST in 1:5000 as a secondary antibody,and TMB(3,3’5,5’-tetramethyl benzidine dihydrochloride)as a shustrate.The specificity and repetitiveness of the ELISA were primely proved by the blocking test.The ELISA was conducted to detect the antisera of mouse and presented good results.Spleen cells of the mice were selected to fuse with NSO cells. The positive wells were selected by indirect ELISA,and after 3 to 4 generations of clone by limited dilution,4 hybridized cell clones(3A3,3G3,4F4,4G3)against Toxoplasma gondii were obtained that the solution titers ranged from 1:800 to 1:12,800,and ascites titers were 1:51200 showing good stability.Identification results of monoclonal antibody subclass was IgG1.The monoclonal ascites were examined with indirect immunofluorescence assay,was against Tachyzoites.Three ascite clones were purified and passed through SDS-PAGE electrophoresis,the result showed that the chain identified by T.gondii Ag strains was approximately 45 kDa.Molecular weights of bands identified by various McAb strains were all over 55kDa.Immunogen was prepared using purified Toxoplasma gondii with the same methods as above,pig had been injected six times with oil-adjuvant-vaccines and liquid-phase-vaccines to produce antibody against Toxoplasma gondii.HRP was used to label pure anti-T. gondii IgG. The mass concentration of IgG was5.73mg·mL-1.McAb 3A3 was selected to develop sandwich ELISA with the HRP-pig-antibody.The best dilution of McAb was 1:100-1:200.The specificity test showed that the method has higher discernment to T. spiraLis ES,C.suis.A. suum,Ⅰ. suis,NPM which belonged to the same family,the OD value of them was remarkable lower than that of Toxoplasma gondii.The establishment of a double decker sandwhich-ELISA provided a simple、fast、sensitive and universally used method for the diagnosis of Toxoplasmosis and Public health,and laid foundations for a rapid diagnostic system for toxoplasmosis.更多还原