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乙型脑炎病毒E蛋白抗原表位E39特异性
单克隆抗体的制备
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华荣虹1,2，3，王斌3，陈娜莎3，于海3，刘天强3，马红2，刘娣1，2，童光志2，3*
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(1.东北林业大学 博士后流动站，黑龙江 哈尔滨150086； 2.黑龙江省农业科学院 畜牧研究中心
博士后工作站，黑龙江 哈尔滨150086； 3.中国农业科学院 哈尔滨兽医研究所 兽医生物技术国家
重点实验室，黑龙江 哈尔滨150001)
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摘要：
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为了制备乙型脑炎病毒E蛋白抗原表位特异性单克隆抗体,将编码乙型脑炎病毒E蛋白抗原表位E39的DNA序列进行人工合成，随后插入表达载体pGEX-6p-1的限制性酶切位点BamHⅠ与XhoⅠ之间，构建了表位肽与GST的融合表达质粒。该表位融合蛋白经亲和层析纯化后免疫BALB/c小鼠，按常规方法进行细胞融合。以化学合成的E39表位多肽为抗原，对融合细胞上清进行间接ELISA筛选。结果，筛选出1株分泌E39表位特异性抗体的杂交瘤细胞株，该杂交瘤细胞分泌抗体能力稳定。经鉴定，该单克隆抗体亚型为IgG2b，轻链类型为κ链。结果表明，用表位融合蛋白为抗原可以制备表位特异性单克隆抗体。 |
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关键词：
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乙型脑炎病毒；E蛋白；单克隆抗体；表位 |
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中图分类号：S 852.659.6 文献标识码：
A 文章编号：1673-4696(2008)02-0133-04 |
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Preparation of monoclonal antibody specific to envelope protein
epitope E39 of Japanese encephalitis virus |
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HUA Rong-hong1,2，3,WANG Bin3,CHEN
Na-sha3,YU Hai3,LIU Tian-qiang3,
MA Hong2,LIU Di1，2,TONG Guang-zhi2，3 |
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(1.Postdoctoral Research Station,Northeast Forest University,Harbin 150086,China;2.Postdoctoral Research Station,Animal Husbandry Research Center,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China；3.National
Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,Chinese Academy of Agricultural
Sciences,Harbin 150001,China) |
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Abstract: The DNA sequence encoding envelope protein epitope E39 of Japanese encephalitis virus was synthesized and inserted into prokaryotic expression vector pGEX-6p-1 between sites BamHⅠ and XhoⅠ.BALB/c mice were immunized with the purified epitope fusion protein GST-E39 expressed in Escherichia coli BL21.The myeloma cells SP2/0 were fused with splenocyte of the immunized BALB/c mice.Hybridoma cell lines were screened and selected by an indirect ELISA using the synthesized peptide E39 as antigen.One hybridoma cell line 5G7 was identified and selected for stably secreting epitope E39-specific antibody.The monoclonal antibody was identified to be IgG2b subtype,and its light chain was κ chain.The results confirmed that the epitope-specific monoclonal antibody could be successfully prepared with the epitope fusion protein as antigen. |
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Key words: Japanese encephalitis virus;envelope protein;monoclonal antibody;epitope |
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