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作 者:王彬[1] 李晓齐[1] 曹红[1] 陈福勇[1] 王永强[1] 郑世军[1]
机构地区:[1]中国农业大学动物医学院农业生物技术国家重点实验室,北京海淀100193
出 处:《中国兽医杂志》2015年第11期23-26,29,共5页Chinese Journal of Veterinary Medicine
基 金:国家自然科学基金(#31272543);现代农业产业技术体系建设专项资金(#NYCYTX-41)
摘 要:为制备能够区分禽白血病A亚群和J亚群病毒的单克隆抗体,将J亚群病毒gp85基因构建到原核表达载体上,并在大肠杆菌BL21中表达携带His标签的禽白血病J亚群GP85融合蛋白,用复性纯化的融合蛋白作为免疫原免疫BALB/c小鼠。4次免疫后取免疫小鼠的脾细胞与骨髓瘤细胞(SP2/0)进行融合,经过3次亚克隆后获得1株稳定分泌针对GP85蛋白的杂交瘤细胞株,命名为386。经间接ELISA测定,小鼠腹水效价为6.4×10~5,亲和力解离常数(kD)为2.18×10^(-9),这株单抗的亚型为IgGl。通过Western Blot和IFA实验证实该株单抗是针对J亚群病毒蛋白GP85的特异性抗体。初步确定此株单克隆抗体的抗原识别区位于GP85蛋白N端的1-50位氨基酸。该株单抗的制备为ALV-J病毒抗原检测以及致病机理的研究奠定了基础。To develop monoclonal antibodies(McAbs) that can distinguish avian leukemia subgroup A virus from subgroup J virus,we cloned gp85 gene into prokaryotic expression vectors pET-32 a and pGEX-6P-1.The expression vectors were transformed into E.coli BL21 for expression of GP85.The purified and refolded fusion protein GP85-His was used as an immunogen to vaccinate BALB/c mice.Then,we fused myeloma cells SP2/0 with splenocytes from the immunized BALB/c mice after four times of immunization.After three rounds of subcloning,we obtained a hybridoma cell clone that stably secreted McAbs against GP85 and named 3B6.Antibody titers were 6.4×10^5 as examined by indirect ELISA in ascite fluid of mice inoculated intraperitoneally with monoclonal antibody-producing hybridoma cells.The dissociation constant(kD) of the monoclonal antibody was determined to be2.18×10^-9 and the isotype was IgG1.Specific recognition of GP85 by anti-GP85 monoclonal antibody was validated by Western Blot and indirect Fluorescence Antibody assay.The epitope in GP85 that was recognized by 3B6 was located between 1 to 50 aa as demonstrated by Western Blot.The McAb will be helpful to the examination of ALV-J antigen and to elucidate the pathogenesis of ALV.
分 类 号:S852.659.3[农业科学—基础兽医学]
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