| 稳定表达犬细小病毒VP2结构蛋白的CHO-K1细胞系的建立 |
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| 引用本文： | 王微,李秀锦,王幸兴,韩冬梅,潘素敏,仲飞.稳定表达犬细小病毒VP2结构蛋白的CHO-K1细胞系的建立[J].中国兽医学报,2010,30(5). |
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| 作者姓名： | 王微 李秀锦 王幸兴 韩冬梅 潘素敏 仲飞 |
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| 作者单位： | 河北农业大学动物科技学院基础兽医系,河北保定,071001 |
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| 基金项目： | 国家自然科学基金资助项目(30771586); 河北省自然科学基金资助项目(C2008000244); 河北省人事厅留学人员科技活动择优资助项目(20080808) |
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| 摘 要： | 为构建犬细小病毒VP2基因分泌性表达细胞系,通过酶切将人CD5信号肽序列从质粒中切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法扩增犬细小病毒VP2基因,并将其插入到pcDNA3.1-CD5sp载体中CD5信号肽的下游,使其与CD5信号肽序列融合,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经脂质体介导转染细胞,后通过G418筛选,建立出稳定表达VP2蛋白的CHO-K1细胞系。测序结果表明,构建的犬细小病毒VP2基因的分泌型表达载体结构正确,表达载体经脂质体介导转染CHO-K1细胞,通过G418加压,筛选出稳定转染VP2基因的细胞株,经PCR检测证明VP2基因已经整合到细胞的染色体中;经RT-PCR、Westernblot分别检测VP2基因表达的mRNA和VP2蛋白,证明犬细小病毒VP2基因能够在CHO-K1细胞进行稳定性表达。这为下一步研究犬细小病毒VP2蛋白与宿主细胞的相互作用及VP2DNA疫苗奠定了基础。
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| 关 键 词： | 犬细小病毒 VP2蛋白 CHO-K1细胞 稳定表达 |
Establishment of CHO-K1 cell lines stably expressing canine parvovirus VP2 structure protein |
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| WANG Wei,LI Xiu-jin,WANG Xing-xing,HAN Dong-mei,PAN Su-min,ZHONG Fei.Establishment of CHO-K1 cell lines stably expressing canine parvovirus VP2 structure protein[J].Chinese Journal of Veterinary Science,2010,30(5). |
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| Authors: | WANG Wei LI Xiu-jin WANG Xing-xing HAN Dong-mei PAN Su-min ZHONG Fei |
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| Institution: | WANG Wei,LI Xiu-jin,WANG Xing-xing,HAN Dong-mei,PAN Su-min,ZHONG Fei (Department of Basic Veterinary Medicine,College of Animal Science , Technology,Agricultural University of Hebei,Baoding,Hebei 071001,China) |
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| Abstract: | In order to construct cell lines stably expressing canine parvovirus VP2 gene,the CD5 signal peptide fragment was transfered from plasmid containing human CD5 signal peptide sequence into multiple clone site of eucaryotic expression vector pcDNA3.1A to generate pcDNA3.1-CD5sp vector.The canine parvovirus VP2 gene amplified by PCR was inserted into pcDNA3.1-CD5sp vector down stream of CD5 signal sequence.The recombinant pcDNA-CD5sp-VP2/MH plasmid was transfected into cells mediated by liposome.The positive c... |
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| Keywords: | canine parvovirus VP2 protein CHO-K1 cell line stable expression |
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