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Volume 16 Issue 2
April 2001

    FANG Liu—rang, CHEN Huan—chun, XIAO Shao—bo, MA Xiang—ru and WANG fei. Cloning，Sequence Analysis and Expression in E ．coli of the EP0 Gene of Pseudorabies Virus Ea Strain[J]. Virologica Sinica, 2001, 16(2): 183-187.
    Citation: FANG Liu—rang, CHEN Huan—chun, XIAO Shao—bo, MA Xiang—ru, WANG fei. Cloning，Sequence Analysis and Expression in E ．coli of the EP0 Gene of Pseudorabies Virus Ea Strain .VIROLOGICA SINICA, 2001, 16(2) : 183-187.
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    伪狂犬病病毒Ea株EP0基因的克隆序列分析 及其在大肠杆菌中的表达

    • 1.

      华中农业太学牧医学院动物病毒室．湖北武汉43007

    • 曲狂犬病病毒Ea株(PRV-Ea)细胞感染物为模板，PCR扩增出1．23 kb的EPO基因完整编码区片段．将该 基因片段克隆到pBluescriptⅡsk+中并构建三个测序质粒，双脱氧末端终止法序列测定并同国外InFh株进行同源 比较，发现PRV-EaEP0基因存在多处点突变和一处缺失突变，但无插^突变和移码。进一步将该片段插入到原核 表达载体pET一28a的His—Tag下游，掏建的原棱表达质粒pETEP0在大肠杆菌Bk】(DE3)中获得了高敛表达，sDs PAGE结果显示，表达的蛋白分子量为62 kD，并形成包橱体。Western印迹分析表明，该蛋白带能与Ea株野毒高 免血清发生特异性反应．证实PRV-Ea EP0基固在B l(D )中获得了正确表达，并且具有免疫学活性。为今后探 ^研究该基因的功能奠定了基础。

    Cloning，Sequence Analysis and Expression in E ．coli of the EP0 Gene of Pseudorabies Virus Ea Strain

    • 1.

      Lab of Animal Virohggy，College ofAnimal Science and Veterinary Medecine,Huazhong Agricultural University．Wuhan 430070，China

    • The 1 23 kb DNA fragment encoding the early protein EP0 of pseudombie8 virus(PRV)Ea strain was amplified by PCR technique and doned inm pBluescriptlI sk+．Thiee sequencing plasmids containing the partial fragment of the EPO gene were constructed and the sequences were obtained by ganger’S sequencing technique Cc~npared wi山PRV InFh strain．there were multipile site-muratiens and a deleted-mutation in the EP0 gene of PRV strain Ea，and the diversity-of anlino acid residu also existed，Then，the EP0 gene was in— serted into an expression vector，pET-28a，fused into the downstream of the 6xHis-Tag in fram e，to yidd the expression plasmid pETEP0 After induction by 球TG．a high expression of fusion protein was obtained． PAGE analysis and Western blotting showed that the fusion protein WaS 62kD an d the protein WaS specific toantisera against PRVEa strain si~dicated thatthe EP0 germ be expressedin l( )andthe ex— pression products have immtmo-genicity
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      Cloning，Sequence Analysis and Expression in E ．coli of the EP0 Gene of Pseudorabies Virus Ea Strain

      • 1. Lab of Animal Virohggy，College ofAnimal Science and Veterinary Medecine,Huazhong Agricultural University．Wuhan 430070，China

      Abstract: The 1 23 kb DNA fragment encoding the early protein EP0 of pseudombie8 virus(PRV)Ea strain was amplified by PCR technique and doned inm pBluescriptlI sk+．Thiee sequencing plasmids containing the partial fragment of the EPO gene were constructed and the sequences were obtained by ganger’S sequencing technique Cc~npared wi山PRV InFh strain．there were multipile site-muratiens and a deleted-mutation in the EP0 gene of PRV strain Ea，and the diversity-of anlino acid residu also existed，Then，the EP0 gene was in— serted into an expression vector，pET-28a，fused into the downstream of the 6xHis-Tag in fram e，to yidd the expression plasmid pETEP0 After induction by 球TG．a high expression of fusion protein was obtained． PAGE analysis and Western blotting showed that the fusion protein WaS 62kD an d the protein WaS specific toantisera against PRVEa strain si~dicated thatthe EP0 germ be expressedin l( )andthe ex— pression products have immtmo-genicity

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