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Volume 18 Issue 2
April 2003

    YU Xiao—lan, XIAO Shao—bo, FANG Liu—rong, JIN Mei—lin and CHEN Huan—chun*. 余晓岚， 肖少波，方六荣，金梅林，陈焕春**[J]. Virologica Sinica, 2003, 18(2): 159-163.
    Citation: YU Xiao—lan, XIAO Shao—bo, FANG Liu—rong, JIN Mei—lin, CHEN Huan—chun*. 余晓岚， 肖少波，方六荣，金梅林，陈焕春** .VIROLOGICA SINICA, 2003, 18(2) : 159-163.
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    牛疱疹病毒I型ul49(VP22)基因的序列分析及其表达

    • 1.

      华中农业大学畜牧兽医学院，湖北武汉，430070

    • 以牛疱疹病毒I型(BHV—I)国内地方分离株感染的细胞培养物制备PCR模板，扩增出大小约O．77kb的 ul49基因完整编码区片段，将扩增片段克隆到pMD18一T载体中，获得含ul49基因的重组质粒pMD—VP22。采用 双脱氧末端终止法进行序列测定，发现与国外Cooper株ul49基因序列完全一致，说明ul49基因相当保守。进一 步将BHV—I ul49完整编码区片段插入原核表达载体pET-28a和真核表达载体pEGFP-C1中，获得分别与6xHis融 合的原核表达质粒pET一28aVP22和与EGFP融合的真核表达质粒pEGFPVP22。将pET一28aVP22转化大肠杆菌 BL21(DE3)，经IPTG诱导，SDS—PAGE电泳检测发现在38kDa处有一条特异性的表达带。利用脂质体介导， 将pEGFPVP22转染PK一15细胞，经G418加压筛选，获得稳定表达VP22的细胞克隆。在倒置荧光显微镜直接检 测未固定的活细胞，发现pEGFPVP22转染细胞能发出很强的荧光并主要集中在细胞核中，而对照载体pEGFP．C1 转染细胞的荧光分布于胞浆。

    余晓岚， 肖少波，方六荣，金梅林，陈焕春**

    • 1.

      Animal Science and Veterinary Medicine College，Huazhong Agricultural University,Wuhan，430070，China

    • A 0．77kb UL49 gene encoding the tegument protein VP22 was amplified from cells culture infected with Bovine herpesvirus 1 (BHV—I)bv PCR．The DNA fragment was further cloned into pM D 1 8一T vector,resulting in recombinant plasmid pM D—VP22．The sequences were determined an d there is no diference with the sequences of the 【，L 9 gene of BHV—I Cooper strain ．Th e 0．77kb fragment was released from plasmid pMD．．VP22 an d subcloned into the vectors pET．．28a an d pEGFP-C 1 respectively，to genera~ a prokaryotic expression pET一28aVP22 an d eukaryotic expression plasmid pEGFPVP22．pET一28aVP22 was consquenfly transformed into E．coli BL21(DE3)．After induced with IPTG．the fusion protein was expressed an d the molecular weight was about 38l(Da． pEGFPVP22 Was tran sfected into PK一15 cells an d the clone cells were selected out wim G418．Th e autofluorescence of the clone cells line tran sfected with pEGFPVP22 could be observed under inve~ed fluorescence microscope．It is interesting that the fluorescence primarily targeted the nucleus of cells whereas the autofluorescence of cells tran sfected with control vector pEGFP—C1 localized throughout the cytoplasm．
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        沈阳化工大学材料科学与工程学院 沈阳 110142

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      余晓岚， 肖少波，方六荣，金梅林，陈焕春**

      • 1. Animal Science and Veterinary Medicine College，Huazhong Agricultural University,Wuhan，430070，China

      Abstract: A 0．77kb UL49 gene encoding the tegument protein VP22 was amplified from cells culture infected with Bovine herpesvirus 1 (BHV—I)bv PCR．The DNA fragment was further cloned into pM D 1 8一T vector,resulting in recombinant plasmid pM D—VP22．The sequences were determined an d there is no diference with the sequences of the 【，L 9 gene of BHV—I Cooper strain ．Th e 0．77kb fragment was released from plasmid pMD．．VP22 an d subcloned into the vectors pET．．28a an d pEGFP-C 1 respectively，to genera~ a prokaryotic expression pET一28aVP22 an d eukaryotic expression plasmid pEGFPVP22．pET一28aVP22 was consquenfly transformed into E．coli BL21(DE3)．After induced with IPTG．the fusion protein was expressed an d the molecular weight was about 38l(Da． pEGFPVP22 Was tran sfected into PK一15 cells an d the clone cells were selected out wim G418．Th e autofluorescence of the clone cells line tran sfected with pEGFPVP22 could be observed under inve~ed fluorescence microscope．It is interesting that the fluorescence primarily targeted the nucleus of cells whereas the autofluorescence of cells tran sfected with control vector pEGFP—C1 localized throughout the cytoplasm．

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