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牛病毒性腹泻病毒p80基因的分段表达
及其抗原性分析
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王丹娜，吴明福，吴立君，王君伟
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（东北农业大学 动物医学院，黑龙江 哈尔滨150030）
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摘要：
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为建立以重组p80蛋白为抗原的牛病毒性腹泻(BVD)鉴别诊断ELISA方法，采用PCR方法克隆了BVDV p80基因的A、B、C三段基因片段，分别连接到原核表达载体pGEX-6P1上，获得了重组质粒pGEX-6P1-p80A、pGEX-6P1-p80B和pGEX-6P1-p80C，将其分别转化感受态菌Rosetta和BL21，经1.0mmol/L的IPTG诱导，分别得到大小为60、47和51ku的目的蛋白。经Western-blotting分析，3个目的蛋白中，只有p80B(289~477aa)基因片段所表达的融合蛋白能与BVDV阳性血清反应，表明p80蛋白的免疫优势区域集中在此部位。该融合蛋白可以作为诊断抗原用于建立ELISA诊断方法。 |
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关键词：
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牛病毒性腹泻病毒；p80基因；表达；抗原性分析 |
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中图分类号：S 852.659.6：Q 786 文献标识码：
A 文章编号：1673-4696(2006)04-0257-05 |
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Expression of overlapping fragments of BVDV p80 gene
and analysis of antigenicity of expressed protein |
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WANG Dan-na, WU Ming-fu, WU Li-jun, WANG Jun-wei |
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(College of Veterinary Medicine，Northeast Agricultural University, Harbin 150030, China)
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Abstract: Three overlapping fragments A, B and C of p80 gene of bovine viral diarrhea virus(BVDV) were amplified by PCR. PCR products were cloned into the expression vector pGEX-6P1 and the recombinant plasmids pGEX-6P1-p80A, pGEX-6P1-p80B and pGEX-6P1-p80C were transformed into E.coli Rosetta and BL21 cells. The target proteins of 60ku, 47ku and 51ku were produced by induction using IPTG at 1.0mmol/L. Only the p80B protein(289-477aa) reacted with BVDV positive serum in Western-blotting, indicating that an immunodominant region of the p80 protein was defined by the p80B protein. The p80B recombinant fusion protein can be used as the specific diagnosis antigen for ELISA assay. |
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Key words: bovine viral diarrhea virus; p80 gene; expression; analysis of antigenicity |
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