B亚群禽白血病病毒单克隆抗体的筛选及应用
发布时间：2018-07-24 14:16
【摘要】：禽白血病对我国养禽业产生了巨大危害,地方品系中A/B亚群禽白血病造成了很大的经济损失,而A/B亚群不易区分,希望能制备出区分ALV-A与ALV-B的单抗,建立一种针对A/B亚群的检测试剂盒,对A/B亚群的净化和防制具有重大意义。利用本实验室制备的针对ALV-B的阳性杂交瘤细胞,对其进一步亚克隆筛选,共进行了4次亚克隆实验,利用A/B亚群禽白血病病毒抗体检测试剂盒,使用羊抗鼠的酶标二抗进行筛选。然后对所筛选的阳性杂交瘤细胞株产生的单抗进行IFA分析。对筛选的单抗进行分型鉴定,采用双抗体夹心法检测亚型。同时对单抗的应用进行了探索,将产生的单抗与ALV-B进行中和,在体外中和试验中,病毒与单抗中和后接种CEF细胞,用ELISA方法检测P27。在体内中和试验中,设置病毒抗体中和组以及攻毒组。单抗稀释后与病毒中和,之后感染实验鸡;攻毒组注射ALV-B的病毒液,3周后取攻毒组的鸡饲喂几种抗病毒药物与单抗进行比较。然后比较分析单抗中和病毒后,对鸡各项指标的影响,共研究了ALV-B抗原的变化、ALV-B抗体的变化、鸡的生长性能变化、免疫器官变化、病理组织的变化、血液指标的变化、以及ALV-B的PCR鉴定。对阳性杂交瘤细胞培养显示其能够稳定分泌单抗,抗体效价为24,亚克隆筛选共筛选了2株A9和G5,在对两株单抗进行IFA检测中发现,G5对ALV-A、ALV-B有荧光反应;A9对ALV-A、ALV-B、ALV-J有荧光反应。这两株单抗都是IgM型。ALV-B与单抗中和后,P27检测为阴性,显示单抗有中和作用。ALV-B与单抗中和后接种动物,3周前并不能检测出抗原阳性,但ALV-AB抗体阳性率很高达83%,在第四周可以检测出P27,抗原阳性率最高达25%,攻毒组抗原阳性率最高达44%,表明病毒抗体中和后在体内能产生较高的抗体阳性率,与攻毒组比较显示抗原阳性率降低。抗体中和组鸡群的生长性能体重在第四周显著高于对照组,攻毒组与对照组在第5周时差异显著,攻毒组体重增重下降将明显。免疫器官指数的变化中,攻毒组胸腺、脾脏、法氏囊在第3-7周内一直高于对照组和抗体中和组,略微肿大;抗体中和组与对照组免疫器官指数在第3-7周基本一致。对抗体中和组鸡进行解剖观察,发现没有明显的病变,在攻毒组一只鸡的组织切片出现淋巴巴细胞样瘤细胞和髓样瘤细胞形成的肿瘤灶,在肝脏中可发现淋巴细胞肿瘤灶,周围肝细胞变性坏死。抗体中和组与对照组的血液各项指标在检测的3周内变化相似,攻毒组的白细胞、淋巴细胞数明显低于抗体中和组与对照组;其它抗病毒药物对攻毒组鸡群血常规检测各项指标明显偏低。提取cDNA,PCR结果结果表明与ALV-B的同源性最高,鉴定为B亚群病毒株。综上所述:阳性杂交瘤细胞株能稳定分泌单抗,亚克隆筛选的单抗能与ALV-B特异性结合但不能区分A/B亚群;单抗与病毒中和后实验,鸡群还是能感染ALV-B但感染率要比攻毒组低,鸡群的生长特性要优于攻毒组,说明单抗中和病毒对鸡群具有一定的免疫保护作用。
[Abstract]:Avian leukosis has caused great harm to poultry industry in China. The A/B subgroup of avian leukemia in local strains has caused great economic loss, and the A/B subgroup is not easy to distinguish. It is hoped to be able to prepare a monoclonal antibody to distinguish between ALV-A and ALV-B, and to establish a detection kit for the A/B subgroup, which is of great significance to the purification and control of A/B subgroups. The positive hybridoma cells prepared for ALV-B were screened for its further subcloning, and 4 subcloning experiments were carried out. The A/B subgroup of avian leukemic virus antibody detection kit was used to screen the antibody of the Sheep anti mouse enzyme labeled two. Then the monoclonal antibody produced by the screened positive hybridoma cell line was analyzed by IFA. The monoclonal antibody was identified and the subtype was detected by the double antibody sandwich method. At the same time, the application of monoclonal antibody was explored. The monoclonal antibody was neutralized with ALV-B. In the neutralization test, the virus and the monoclonal antibody were neutralized and inoculated with CEF cells, and the ELISA method was used to detect the neutralization and attack of the virus antibody in the body and in the experiment. After the monoclonal antibody was diluted with the virus and then infected with the experimental chicken, the attack group was injected with ALV-B virus. After 3 weeks, the chickens were fed with several antiviral drugs and compared with the McAbs. Then the changes of ALV-B antigen, the change of ALV-B antibody and the growth of chicken were studied. Performance changes, changes in immune organs, changes in pathological tissue, changes in blood indexes, and PCR identification of ALV-B. The positive hybridoma cell culture showed that it was able to secrete monoclonal antibody steadily, the titer of antibody was 24, and 2 A9 and G5 were screened by subclone screening, and G5 was found to have fluorescent reaction to ALV-A, ALV-B in two monoclonal antibodies; A9 ALV-A, ALV-B, ALV-J have fluorescence reaction. The two mAbs are all neutralization of IgM type.ALV-B and mAb, P27 detection is negative. It shows that mAb has neutralization action of.ALV-B and mAb and inoculated animals. The antigen positive rate can not be detected before 3 weeks, but the positive rate of ALV-AB antibody is up to 83%, and the positive rate of antigen can be detected in the fourth week. The positive rate of antigen is the highest The positive rate of antigen in the attack group was up to 44%, which showed that the antibody positive rate of the virus antibody neutralized in the body was higher than that of the attack group. The growth performance weight of the antibody neutralization group was significantly higher in the fourth week than the control group. The difference between the attack group and the control group was significant at fifth weeks, and the weight of the attack group was in the attack group. The weight of the attack group and the control group were significantly higher than the control group at fifth weeks. In the changes of the immune organ index, the thymus, spleen and bursa of the attack group had been higher than the control group and the antibody neutralization group in 3-7 weeks, and the immune organ index of the antibody neutralization group and the control group were basically the same. In the tissue section of a chicken, lymphoblastic and myeloid tumor cells were found in a chicken tissue section. Lymphocyte tumors were found in the liver and the peripheral hepatocytes were necrotic. The blood indexes of the antibody neutralization group and the control group were similar in the 3 weeks. The number of lymphocytes in the attack group was significantly lower. In the antibody neutralization group and the control group, the other antiviral drugs were obviously lower in the blood routine detection of the chicken group in the attack group. The results of cDNA, PCR results showed that the homology of the ALV-B was the highest, and the B subgroup virus was identified. The positive hybridoma cell line could stabilize the secretory monoclonal antibody and the monoclonal antibody of the subclone screening was specific to the ALV-B specificity The A/B subgroup can not be distinguished, but the chicken group can infect ALV-B but the infection rate is lower than that of the attack group. The growth characteristic of the chicken group is better than that of the attack group, which shows that the monoclonal antibody neutralization virus has a certain immune protective effect on the chicken group.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：2141638
[Abstract]:Avian leukosis has caused great harm to poultry industry in China. The A/B subgroup of avian leukemia in local strains has caused great economic loss, and the A/B subgroup is not easy to distinguish. It is hoped to be able to prepare a monoclonal antibody to distinguish between ALV-A and ALV-B, and to establish a detection kit for the A/B subgroup, which is of great significance to the purification and control of A/B subgroups. The positive hybridoma cells prepared for ALV-B were screened for its further subcloning, and 4 subcloning experiments were carried out. The A/B subgroup of avian leukemic virus antibody detection kit was used to screen the antibody of the Sheep anti mouse enzyme labeled two. Then the monoclonal antibody produced by the screened positive hybridoma cell line was analyzed by IFA. The monoclonal antibody was identified and the subtype was detected by the double antibody sandwich method. At the same time, the application of monoclonal antibody was explored. The monoclonal antibody was neutralized with ALV-B. In the neutralization test, the virus and the monoclonal antibody were neutralized and inoculated with CEF cells, and the ELISA method was used to detect the neutralization and attack of the virus antibody in the body and in the experiment. After the monoclonal antibody was diluted with the virus and then infected with the experimental chicken, the attack group was injected with ALV-B virus. After 3 weeks, the chickens were fed with several antiviral drugs and compared with the McAbs. Then the changes of ALV-B antigen, the change of ALV-B antibody and the growth of chicken were studied. Performance changes, changes in immune organs, changes in pathological tissue, changes in blood indexes, and PCR identification of ALV-B. The positive hybridoma cell culture showed that it was able to secrete monoclonal antibody steadily, the titer of antibody was 24, and 2 A9 and G5 were screened by subclone screening, and G5 was found to have fluorescent reaction to ALV-A, ALV-B in two monoclonal antibodies; A9 ALV-A, ALV-B, ALV-J have fluorescence reaction. The two mAbs are all neutralization of IgM type.ALV-B and mAb, P27 detection is negative. It shows that mAb has neutralization action of.ALV-B and mAb and inoculated animals. The antigen positive rate can not be detected before 3 weeks, but the positive rate of ALV-AB antibody is up to 83%, and the positive rate of antigen can be detected in the fourth week. The positive rate of antigen is the highest The positive rate of antigen in the attack group was up to 44%, which showed that the antibody positive rate of the virus antibody neutralized in the body was higher than that of the attack group. The growth performance weight of the antibody neutralization group was significantly higher in the fourth week than the control group. The difference between the attack group and the control group was significant at fifth weeks, and the weight of the attack group was in the attack group. The weight of the attack group and the control group were significantly higher than the control group at fifth weeks. In the changes of the immune organ index, the thymus, spleen and bursa of the attack group had been higher than the control group and the antibody neutralization group in 3-7 weeks, and the immune organ index of the antibody neutralization group and the control group were basically the same. In the tissue section of a chicken, lymphoblastic and myeloid tumor cells were found in a chicken tissue section. Lymphocyte tumors were found in the liver and the peripheral hepatocytes were necrotic. The blood indexes of the antibody neutralization group and the control group were similar in the 3 weeks. The number of lymphocytes in the attack group was significantly lower. In the antibody neutralization group and the control group, the other antiviral drugs were obviously lower in the blood routine detection of the chicken group in the attack group. The results of cDNA, PCR results showed that the homology of the ALV-B was the highest, and the B subgroup virus was identified. The positive hybridoma cell line could stabilize the secretory monoclonal antibody and the monoclonal antibody of the subclone screening was specific to the ALV-B specificity The A/B subgroup can not be distinguished, but the chicken group can infect ALV-B but the infection rate is lower than that of the attack group. The growth characteristic of the chicken group is better than that of the attack group, which shows that the monoclonal antibody neutralization virus has a certain immune protective effect on the chicken group.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
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1 陈蕾;顾剑新;;禽白血病的流行病学与检测方法的研究进展[J];安徽农业科学;2010年27期
相关硕士学位论文 前1条
1 王峰;J亚群禽白血病病毒和禽网状内皮组织增生症病毒单克隆抗体的研制[D];山东农业大学;2011年
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