B亞群禽白血病病毒單克隆抗體的篩選及應(yīng)用
發(fā)布時間：2018-07-24 14:16
【摘要】：禽白血病對我國養(yǎng)禽業(yè)產(chǎn)生了巨大危害,地方品系中A/B亞群禽白血病造成了很大的經(jīng)濟(jì)損失,而A/B亞群不易區(qū)分,希望能制備出區(qū)分ALV-A與ALV-B的單抗,建立一種針對A/B亞群的檢測試劑盒,對A/B亞群的凈化和防制具有重大意義。利用本實驗室制備的針對ALV-B的陽性雜交瘤細(xì)胞,對其進(jìn)一步亞克隆篩選,共進(jìn)行了4次亞克隆實驗,利用A/B亞群禽白血病病毒抗體檢測試劑盒,使用羊抗鼠的酶標(biāo)二抗進(jìn)行篩選。然后對所篩選的陽性雜交瘤細(xì)胞株產(chǎn)生的單抗進(jìn)行IFA分析。對篩選的單抗進(jìn)行分型鑒定,采用雙抗體夾心法檢測亞型。同時對單抗的應(yīng)用進(jìn)行了探索,將產(chǎn)生的單抗與ALV-B進(jìn)行中和,在體外中和試驗中,病毒與單抗中和后接種CEF細(xì)胞,用ELISA方法檢測P27。在體內(nèi)中和試驗中,設(shè)置病毒抗體中和組以及攻毒組。單抗稀釋后與病毒中和,之后感染實驗雞;攻毒組注射ALV-B的病毒液,3周后取攻毒組的雞飼喂幾種抗病毒藥物與單抗進(jìn)行比較。然后比較分析單抗中和病毒后,對雞各項指標(biāo)的影響,共研究了ALV-B抗原的變化、ALV-B抗體的變化、雞的生長性能變化、免疫器官變化、病理組織的變化、血液指標(biāo)的變化、以及ALV-B的PCR鑒定。對陽性雜交瘤細(xì)胞培養(yǎng)顯示其能夠穩(wěn)定分泌單抗,抗體效價為24,亞克隆篩選共篩選了2株A9和G5,在對兩株單抗進(jìn)行IFA檢測中發(fā)現(xiàn),G5對ALV-A、ALV-B有熒光反應(yīng);A9對ALV-A、ALV-B、ALV-J有熒光反應(yīng)。這兩株單抗都是IgM型。ALV-B與單抗中和后,P27檢測為陰性,顯示單抗有中和作用。ALV-B與單抗中和后接種動物,3周前并不能檢測出抗原陽性,但ALV-AB抗體陽性率很高達(dá)83%,在第四周可以檢測出P27,抗原陽性率最高達(dá)25%,攻毒組抗原陽性率最高達(dá)44%,表明病毒抗體中和后在體內(nèi)能產(chǎn)生較高的抗體陽性率,與攻毒組比較顯示抗原陽性率降低�？贵w中和組雞群的生長性能體重在第四周顯著高于對照組,攻毒組與對照組在第5周時差異顯著,攻毒組體重增重下降將明顯。免疫器官指數(shù)的變化中,攻毒組胸腺、脾臟、法氏囊在第3-7周內(nèi)一直高于對照組和抗體中和組,略微腫大;抗體中和組與對照組免疫器官指數(shù)在第3-7周基本一致。對抗體中和組雞進(jìn)行解剖觀察,發(fā)現(xiàn)沒有明顯的病變,在攻毒組一只雞的組織切片出現(xiàn)淋巴巴細(xì)胞樣瘤細(xì)胞和髓樣瘤細(xì)胞形成的腫瘤灶,在肝臟中可發(fā)現(xiàn)淋巴細(xì)胞腫瘤灶,周圍肝細(xì)胞變性壞死�？贵w中和組與對照組的血液各項指標(biāo)在檢測的3周內(nèi)變化相似,攻毒組的白細(xì)胞、淋巴細(xì)胞數(shù)明顯低于抗體中和組與對照組;其它抗病毒藥物對攻毒組雞群血常規(guī)檢測各項指標(biāo)明顯偏低。提取cDNA,PCR結(jié)果結(jié)果表明與ALV-B的同源性最高,鑒定為B亞群病毒株。綜上所述:陽性雜交瘤細(xì)胞株能穩(wěn)定分泌單抗,亞克隆篩選的單抗能與ALV-B特異性結(jié)合但不能區(qū)分A/B亞群;單抗與病毒中和后實驗,雞群還是能感染ALV-B但感染率要比攻毒組低,雞群的生長特性要優(yōu)于攻毒組,說明單抗中和病毒對雞群具有一定的免疫保護(hù)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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
本文編號：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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳蕾;顧劍新;;禽白血病的流行病學(xué)與檢測方法的研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2010年27期
相關(guān)碩士學(xué)位論文 前1條
1 王峰;J亞群禽白血病病毒和禽網(wǎng)狀內(nèi)皮組織增生癥病毒單克隆抗體的研制[D];山東農(nóng)業(yè)大學(xué);2011年
，本文編號：2141638
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