| 犬细小病毒结构蛋白VP2基因的克隆和在毕赤酵母中表达 |
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| 引用本文： | 贺英,史秋梅,潘素敏,张艳英,邵欣华.犬细小病毒结构蛋白VP2基因的克隆和在毕赤酵母中表达[J].兽医大学学报,2013(11):1642-1646. |
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| 作者姓名： | 贺英 史秋梅 潘素敏 张艳英 邵欣华 |
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| 作者单位： | [1]河北科技师范学院动物科技学院河北省预防兽医重点实验室,河北昌黎066600 [2]河北新华科极兽药有限公司,河北石家庄051430 |
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| 基金项目： | 石家庄市科技支撑计划项目（09150293A）;河北科技师范学院科研创新团队项目（CXTD2012-01）;河北省自然科学基金资助项目（C2013407106） |
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| 摘 要： | 表达犬细小病毒VP2蛋白（CPV—VP2），用于重组蛋白免疫小鼠制备单克隆抗体，并为犬细小病毒病的诊断奠定基础。采用PCR方法对CPVVP2基因进行扩增，将CPVVP2基因克隆到毕赤酵母分泌表达载体pPICZAA中，构建真核重组表达载体pPICZAA-VP2，将该重组质粒线性化后，转化巴斯德毕赤酵母（Pichiapastoris）X-33中，甲醇诱导表达CPVVP2，SDs_PAGE和Westernblotting鉴定表达蛋白。结果，成功扩增了CPV—VP2基因，构建了真核重组表达载体pPICZAA—VP2，在毕赤酵母菌中表达出约68000蛋白。Western—blotting鉴定表明，表达蛋白为目的蛋白VP2。表达菌株扩大表达体系于培养基中培养，上清液用70％过硫酸铵4℃沉淀浓缩，采用His选择镍-亲和层析柱分离纯化获得重组的酵母表达的带组氨酸标签的VP2蛋白，表达量约3mg／L。结果表明，在毕赤酵母中成功地表达了CPV—VP2蛋白，且能被犬细小病毒VP2单克隆抗体特异识别。
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| 关 键 词： | 犬细小病毒VP2基因 毕赤酵母 真核表达 |
Cloning and expression of VP2 gene of canine parvovirus in Pichia pastoris |
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| Authors: | HE Ying SHI Qiu-mei PAN Su-min ZHANG Yan-ying SHAO Xin-hua |
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| Institution: | z ( 1. Department of Animal Science, Hebei Key Laboratory of Preventive Veterinary, Hebei Normal University of Science and Technology ,Changli, Hebei 066600, China ; 2. Animal medicine In Company of He- bei Xinhua Kej i, Shij iazhuang 051430, China) |
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| Abstract: | CPV-VP2 gene was amplified by PCR, cloned into Pichia pastoris expression vector pPICZAA,and an eukaryotic expression vector was constructed , named pPICZAA-VP21. The vector was lineazed and transformed into Pichia pastoris X-33 by electroporation method. The protein expression were induced in medium with methanol, SDS- PAGE and Western blotting a- nalysis of the culture supernatants confirmed that the VP2 protein of 68kD was expressed in Pichia pastoris. The secreted VP2 was concentrated from BMMY medium by 70% ammonium sul- fate precipitation. His-tagged VP2 protein was then isolated at 4℃ on a 20 mL HisSelect nickel- affinity column （Sigma） . HisSelect affinity chromatography yielded approximately 3 mg/L of protein. CPV-VP2 was successfully expressed in yeast Pichia astoris. The expressed CPV-VP2 protein shares reaction to monoclonal antibody against VP2. |
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| Keywords: | CPV-VP2 gene Pichia pastoris eukaryotic expression |
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