(中国农业科学院哈尔滨兽医研究所实验动物与比较医学团队,兽医生物技术国家重点实验室,黑龙江省实验动物与比较医学重点实验室,哈尔滨 150069)
Q95-33
WANG Xingtong
Division of Laboratory Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin 150069, ChinaMENG Xing
Division of Laboratory Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin 150069, ChinaTONG Xianghui
Division of Laboratory Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin 150069, ChinaCHEN Hongyan
Division of Laboratory Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin 150069, ChinaHAN Lingxia
Division of Laboratory Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin 150069, China(Division of Laboratory Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin 150069, China)
目的 制备鸭抗原处理相关转运体(TAP)特异性单抗,为深入利用实验鸭开展免疫学研究提供实验材料?方法 利用大肠埃希菌诱导表达主要组织相容性复合体(MHC)单倍型HBW-SPF 鸭TAP 蛋白肽结合区片段,表达产物经镍柱纯化后免疫BALB/ c 小鼠,采用ELISA 技术筛选特异性单抗分泌杂交瘤细胞株?将阳性细胞株制备小鼠腹水,作为一抗,与多次截短表达TAP 蛋白肽结合区进行蛋白免疫印迹试验,鉴定单抗针对的抗原表位?通过间接免疫荧光试验比较该单抗对实验鸭和鸡外周血淋巴细胞的反应性,利用免疫组织化学技术检测对SPF 鸡?SPF 鸭?鹌鹑?鹅和SPF 猪的特异性?结果 获得一株鸭TAP 单抗1A6,抗原表位位于297NARHQMLQQAVLDATAGTGMVVQEAI322,对鸡和鸭外周血淋巴细胞具有免疫荧光反应性;在鸡和鸭的肠黏膜固有层检测到大量特异性信号,在猪?鹌鹑和鹅没有检测到信号?结论 获得了一株具有鸡和鸭反应性的抗原转运相关体特异性的单抗,可运用于禽类实验动物在禽病学和禽免疫学方面的研究?
Objective A monoclonal antibody (Mab) against transporter associated with antigen processing (TAP)was prepared to study the function of TAP protein in the immunogenetics of ducks. Methods BALB/ c mice were immunizedwith fused Escherichia coli expression products containing peptide-binding region of TAP protein of major histocompatibilitycomplex (MHC) haplotype SPF ducks. Specific monoclonal antibodies were screened by ELISA, and truncated expressionmethod was used to identify the antigenic epitope. An indirect immunofluorescence assay was used to compare the reactivity ofMab against the peripheral blood lymphocytes of ducks and chickens. Immunohistochemistry was used to identify thespecificity of the Mab was detected against SPF chickens, SPF ducks, SPF pigs, common quails and geese. Results A duckTAP-specific monoclonal antibody was obtained and named as “1A6”. The antigenic epitope against 1A6 was identifiedroughly at 297NARHQMLQQAVLDATAGTGMVVQEAI322 by a trimmed expressed fusion protein. Chicken and duck peripheralbloodlymphocytes were tested positive for 1A6 in this indirect immunofluorescence assay. Immunohistochemistry of theduodenum from SPF chickens, SPF ducks, SPF pigs, Corturnix and geese by 1A6 was undertaken. Specific strong signals inimmunohistochemistry assay were detected in the duodenum of chickens and ducks, but not detected in the pigs, quails,geese or negative-control jejunum. Conclusions A monoclonal antibody specific to the TAP protein of chickens and ducks is obtained. It provides a useful material for studies of avian diseases and immunology of poultry.
王兴童,孟兴,佟相慧,陈洪岩,韩凌霞.主要组织相容性复合体单倍型鸭抗原处理相关转运体单抗的制备[J].中国实验动物学报,2019,27(2):200~207.
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