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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (4): 54-62    DOI: 10.13523/j.cb.20180408
技术与方法     
抗树鼩CD3ε单克隆抗体的制备及生物学特性鉴定
徐婧雯,张雪梅,吴忠香,朱文兵,蒋曦,巩蔚,严丽蔚,宋杰,李慧,董少忠()
中国医学科学院/北京协和医学院医学生物学研究所 云南省重大传染病疫苗研发重点实验室实验树鼩标准化与应用研究省创新团队 昆明 650118
Preparation and Identification of Monoclonal Antibodies Against Tree Shrews CD3ε
Jing-wen XU,Xue-mei ZHANG,Zhong-xiang WU,Wen-bing ZHU,Xi JIANG,Wei GONG,Li-wei YAN,Jie SONG,Hui LI,Shao-zhong DONG()
Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease, Yunnan Innovation Team of Standardization and Application Research in Tree Shrew. Kunming 650118, China
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摘要： 目的

制备鼠抗树鼩CD3ε单克隆抗体,并对其生物学特性进行鉴定。

 方法

以GST-CD3ε蛋白为免疫原免疫BALB/c小鼠,利用杂交瘤技术融合免疫后的BALB/c小鼠脾细胞和小鼠骨髓瘤SP2/0细胞,通过间接酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)方法检测,筛选出多株能分泌抗树鼩CD3ε的杂交瘤细胞株,经过3次亚克隆筛选后,制备小鼠腹水单克隆抗体,并纯化得到鼠抗树鼩CD3ε单克隆抗体,通过腹水单克隆抗体效价测定、单克隆抗体亲和力测定、单克隆抗体抗原表位分析、 Western blot和流式细胞荧光分选技术(fluorescence activated cell sorting, FACS)分析其生物学特性。

 结果

筛选到5株能稳定分泌抗树鼩CD3ε单克隆抗体的杂交瘤细胞株,命名为78I、87I、92D1、75II和35C8,腹水单克隆抗体效价分别为1:10 6、1:10 6、1:10 4、1:10 6、1:10 3,亲和力解离常数(Kd)分别为1.8×10 -5、2.9×10 -5、4.9×10 -5、7.3×10 -5、3.6×10 -5。5株单克隆抗体抗原表位分析显示78I、87I和75II识别同一抗原表位,而92D1和35C8识别另一抗原表位。经Western blot检测,HRP标记后的92D1能识别GST-CD3ε蛋白和树鼩外周血单个核细胞(peripheral blood mononuclear cell, PBMC),并对大鼠、小鼠和猴的PBMC有抗体交叉反应。经FACS检测,PE-Cy5.5标记后的92D1能特异性识别树鼩PBMC。

结论

成功制备出鼠抗树鼩CD3ε的单克隆抗体,为进一步应用于树鼩免疫检测奠定基础。
关键词： 树鼩;  CD3ε;  单克隆抗体    
Abstract: Objective:

To develop monoclonal antibodies (mAbs) against tree shrews CD3ε, and identify their biological characteristics.

 Method:

BALB/c mice were immunized with GST-CD3ε protein as an immunogen. Immunized mice spleen cells were fused with SP2/0 cells with the hybridoma technique. GST- CD3ε proteins and GST proteins were used as coating antigens to establish the indirect ELISA. After screening and three rounds of cloning process, the strain of hybridomas secreting anti-CD3ε mAbs were obtained. More anti- CD3ε mAbs were prepared by mice ascites, which were purified by Protein A resin. Then anti- CD3ε mAbs were identified with indirect ELISA, superposable ELISA, antibody titer evaluation, Western blot and FACS.

 Result:

Five hybridomas were obtained, and named 78I, 87I, 92D1, 75II and 35C8.The titer of five ascites were 1:10 6, 1:10 6, 1:10 4, 1:10 6 and 1:10 3. The dissociation constant (Kd) of these ascites were 1.8×10 -5, 2.9×10 -5, 4.9×10 -5, 7.3×10 -5 and 3.6×10 -5. Monoclonal antibody epitope analysis revealed that 78I, 87I, and 75II recognized the same antigenic epitope, whereas 92D1 and 35C8 recognized another antigenic epitope of GST- CD3ε. Western blot analysis showed that HRP-labeled 92D1 recognized GST-CD3ε and tree shrews’ PBMC, and had an antibody cross-reactivity with PBMC from rats, mice and monkeys. After FACS detection, 92D1 labeled with PE-Cy5.5 can specifically identify the PBMC of tree shrews.

Conclusion:

Murine anti-tree shrews’ CD3ε mAbs were successfully prepared, which laid the foundation for the further application in immune detection of tree shrews.
Key words: Tree shrews    CD3ε    Monoclonal antibodies
收稿日期: 2017-12-13 出版日期: 2018-05-08
ZTFLH:  Q813.2  
基金资助: 云南省科技人才和平台计划(2017HC019);云南省科技计划资助项目(2016BC004)
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徐婧雯
张雪梅
吴忠香
朱文兵
蒋曦
巩蔚
严丽蔚
宋杰
李慧
董少忠

引用本文:

徐婧雯,张雪梅,吴忠香,朱文兵,蒋曦,巩蔚,严丽蔚,宋杰,李慧,董少忠. 抗树鼩CD3ε单克隆抗体的制备及生物学特性鉴定[J]. 中国生物工程杂志, 2018, 38(4): 54-62.

Jing-wen XU,Xue-mei ZHANG,Zhong-xiang WU,Wen-bing ZHU,Xi JIANG,Wei GONG,Li-wei YAN,Jie SONG,Hui LI,Shao-zhong DONG. Preparation and Identification of Monoclonal Antibodies Against Tree Shrews CD3ε. China Biotechnology, 2018, 38(4): 54-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180408    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I4/54

Primer name Primer sequence (5'-3')
CD3ε-F CCGGAATTCGAGGACGATAACGACAAAACT
CD3ε-R CCGCTCGAGTCAGATGCCTCTCTGATTCA
表1  重组蛋白原核表达载体构建所用引物序列
图1  基因克隆以及重组质粒双酶切鉴定
图2  重组蛋白GST-CD3ε和标签蛋白GST的表达及纯化
图3  CD3ε腹水单克隆抗体纯化
图4  CD3ε单克隆抗体亲和力测定
mAb 78I 87I 92D1 75II 35C8
OD450 AI(%) OD450 AI(%) OD450 AI(%) OD450 AI(%) OD450 AI(%)
78I 0.92 /
87I 0.92 0% 0.92 /
92D1 2.40 154% 1.51 60% 0.97 /
75II 1.13 40% 1.00 24% 1.92 131% 0.69 /
35C8 2.40 167% 1.73 92% 0.96 4% 1.99 154% 0.88 /
表2  CD3ε单克隆抗体相加ELISA结果
图5  CD3ε单克隆抗体Western-blot鉴定
图6  流式细胞术检测CD3ε单克隆抗体对树鼩PBMC的识别
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