【作者】 刘秋燕；

【导师】 王川庆；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 本研究将纯化的鸡传染性法氏囊病毒和鸡志贺氏菌毒素(LPS)用生理盐水稀释到适当浓度,按1:1的比例混合后,加入特殊免疫增强剂制成混合抗原免疫小鼠。用一次融合、不同抗原包被分别筛选的方法,获得了能分别稳定分泌抗此两种抗原的单克隆抗体杂交瘤细胞株。与用单一纯化抗原免疫小鼠的传统方法相比,本研究建立的混合免疫、分别筛选方法,仅一次免疫、一次融合就可选出分别分泌两种抗不同抗原的单克隆抗体的杂交瘤细胞株,是一种建立杂交瘤细胞株的高效方法,不仅节约了实验材料、降低了成本,而且节省了时间和人力,大大提高了建立杂交瘤细胞株的工作效率。原来需要三个或更多人才能完成的工作,现在一个人就可以完成,这对杂交瘤技术的学术研究和实际应用均具有重要意义。通过阳性杂交瘤细胞的筛选、克隆和亚克隆,最终获得了3株抗He4 IBDV的杂交瘤细胞株4H5、482和3C11,4株抗鸡志贺氏菌毒素LPS的杂交瘤细胞株3C2、3C4、482和4A3。经鉴定其免疫球蛋白均为IgG2。特异性鉴定结果表明抗He4 IBDV的3株单克隆抗体与AIV、NDV、PRV、PRRSV和LPS均无交叉反应,IBDV对其有较好的阻断效果。抗鸡志贺氏菌毒素LPS的4株单克隆抗体与AIV、NDV、PRV、PRRSV和IBDV也无交叉反应,鸡志贺氏菌毒素LPS对其有较好的阻断效果;说明所选的两种杂交瘤细胞株均具有较高的特异性。3株抗He4 IBDV的单克隆抗体杂交瘤细胞株的细胞培养上清和腹水效价分别最高可以达到1:12800和1:51200以上;4株抗LPS的单克隆抗体杂交瘤细胞株的细胞培养上清和腹水效价分别最高可以达到1:51200和1:409600以上。经过连续传代和反复冻融之后,这两种杂交瘤细胞仍能稳定分泌抗体,抗体效价基本稳定,且对酸、碱、热均有良好的稳定性。免疫小鼠后收集的腹水经辛酸硫酸铵纯化后,蛋白含量分别为78.45mg/mL、88.65mg/mL。用所制单抗建立的双夹心ELISA方法检测IBDV,最低检出浓度为390.8ng/mL,敏感性较高。用鸡志贺氏菌毒素LPS进行小鼠攻毒实验,发现单克隆抗体的细胞上清和腹水对其有一定的中和作用。本实验中此两种单克隆抗体的获得为鸡传染性法氏囊病和鸡志贺氏菌病的学术研究及快速诊断方法的建立奠定了基础,具有一定的应用价值,并且本论文建立的生产杂交瘤细胞的方法在单克隆抗体杂交瘤细胞生产技术上是一次大胆尝试和新的突破,具有一定的生产意义。更多还原

【Abstract】 Infectious bursal disease virus and Lipopolysaccharide of Shigellae in the Chicken were maded mixed antigens in this study.Mice was immunized with the mixed antigens,which added Bursin.Hybridoma cell strains secreting monoclonal antibodies against two antigens were obtained by one fusion and screen respectively methods. Compared with traditional method using sole antigen immunize mouse,this method used mix immunity,screening respectively,is a highly effective method in obtained the hybridoma cell strains.Hybridoma cell strains against different antigens can obtained just through one immunity and one fusion.This method not only has saved the experimental material,reduced the cost,moreover,it has saved the time and the manpower,greatly enhanced the working efficiency.Now one people could finish the work needed three or more people finished before.This has the vital significance to the scholarly research and the practical application of hybridlump technology.IBDV and LPS of Shigellae in the Chicken was purified by sucrose concentra-tion and differential centrifugation.Eight-week Balb/c mice were immunized according to Kazuhiro’s produre.Murine splenocytes of the immunized mice after the third immunization were fused with SP2/0 myelomas,An indirect Enzyme-linked Immunosorbent Assay(ELISA)coated with He4 IBDV and LPS was used to screen hybridoma for production of specific antibody in hybridoma culture fluid.Three hybriddoma against Infectious bursal disease virus were generated,named 4H5、4B2、3C11,four hybridoma against Lipopolysaccharide were generated,named 3C2、3C4、4B2、4A3.The monoclonal antibodies(McAbs)were categorized into IgG2 subtype.The result of cross-reaction in ELISA dedicated that seven McAbs were type-sepecific.The indirect ELISA titers of the 4H5 hybridoma against Infectious bursal disease virus in hybridoma culture fluid and mouse ascites were 1:25600 and 1:102400 respectively.The ELISA titers of the 4H5 McAbs in hybridoma culture fluid and mouse ascites were 1:102400 and 1:819200 respectively.The cell strain also can stably secrete the McAbs after twenty-six serial passages and freezing-thawing three times within six months.Mouse ascites was purified and protein concentrations were 78.45mg/mL and 88.65mg/mL respectively.The obtained of the two species hybridoma cell strains against two antigens laied the foundations of study and rapid diagnosis of Infectious bursal disease virus and Shigellae in the Chicken.更多还原