HUANG Wei, XU Ge-lin, HU Qiao-ling, LI Ping, WANG Ji-lin, YAN jia-xing, WU Jie, ZHU Yu-tao, RUAN Li and ZHU Jia-hong. Construction and Immunogenicity Study of the Improved Non-replicating Vacc- inia Recombinant Co-expressing Rabies Virus Glycoprotein and Nucleoprotein[J]. Virologica Sinica, 2004, 19(4): 376-379.
Citation: HUANG Wei, XU Ge-lin, HU Qiao-ling, LI Ping, WANG Ji-lin, YAN jia-xing, WU Jie, ZHU Yu-tao, RUAN Li, ZHU Jia-hong. Construction and Immunogenicity Study of the Improved Non-replicating Vacc- inia Recombinant Co-expressing Rabies Virus Glycoprotein and Nucleoprotein .VIROLOGICA SINICA, 2004, 19(4) : 376-379.

双表达狂犬病毒基因的非复制型痘苗病毒改建及免疫效果*

  • 为减少重组病毒非必需外源基因,进一步提高非复制重组痘苗病毒狂犬病疫苗的安全性,本研究改建不含报道基因LacZ的双表达狂犬病毒aG株G、N的非复制重组痘苗病毒VTKRGΔCKRN。采用G418-neo富集、蓝白斑及免疫蚀斑筛选等方法,以表达狂犬病毒糖蛋白(RG)基因 的非复制重组痘苗病毒VTKRG△CKlacZ作为亲本株,利用同源重组原理,删除C与K片断间lacZ,并将狂犬病毒核蛋白(RN)基因插入C-K片段间。 经核酸及蛋白水平检测表明VTKRGΔCKRN能同时稳定有效地表达狂犬病毒G和N,并具有非复制病毒生长特性。重组病毒裸鼠毒力实验表明VTKRG△CKRN较复制型重组痘苗病毒VTKRG病毒毒力显著减弱。VTKRGΔCKRN免疫小鼠可诱生较高有效中和抗体,并能保护小鼠免于致死剂量狂犬病毒攻击。以1.6×106PFU较低免疫剂量、仅一次免疫狗可保护狗经致死量中国狂犬病毒街毒株SBD株攻击后存活,诱生具有保护性中和抗体。非复制狂犬-痘苗重组病毒VTKRGΔCKRN免疫效果好、更具安全性。

Construction and Immunogenicity Study of the Improved Non-replicating Vacc- inia Recombinant Co-expressing Rabies Virus Glycoprotein and Nucleoprotein

  • Corresponding author: ZHU Jia-hong, 
  • To improve the safety of recombinant vaccinia virus,a non-replicating vaccinia recombi- nant co-expressing rabies virus glycoprotein(RG) and nucleoprotein(RN) without a selectable marker was reconstructed. LacZ between fragment C and fragment K in non-replicating recombinant vaccinia virus VTKRG△CKlacZ was deleted and was substituted with rabies virus nucleoprotein by homologous recombination resulting in VTKRGΔCKRN. The recombinant vaccinia virus was screened by a two-step selection under, G418 and with White/Blue plaque color change as well as immunofluorescence. By Dot Blot analysis, RG gene and RN gene were shown to integrate stably into the non-replicating vaccinia recombinants even after 20 passages in CEF cells. The virulence of VTKRGΔCKRN was obviously less than that of VTKRG in naked mouse. The non-replicating characterization of VTKRGΔCKRN was also stable. It was proved that VTKRGΔCKRN could express RG and RN at the same time. VTKRGΔCKRN could elicit viral neutralizing antibody(VNA) similar to that of VTKRG by inoculated into the mice,and thus protect the animals from lethal challenge of rabies virus. It could elicit efficient viral neutralizing antibody(VNA) and protect the dogs from lethal challenge of rabies street strain SBD by intramuscular inoculation at dose of only 1.6×106PFU. This study confirmed the efficacy and safety of non-replicating recombinant vaccinia virus VTKRGΔCKRN.

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    Construction and Immunogenicity Study of the Improved Non-replicating Vacc- inia Recombinant Co-expressing Rabies Virus Glycoprotein and Nucleoprotein

      Corresponding author: ZHU Jia-hong,
    • 1. 1.Wuhan Institute of Biological Products, Wuhan 430060,China
    • 2. Institute for Viral Disease Control and Prevention, The Chinese Centre for Disease Control and Prevention , Beijing 100050,China

    Abstract: To improve the safety of recombinant vaccinia virus,a non-replicating vaccinia recombi- nant co-expressing rabies virus glycoprotein(RG) and nucleoprotein(RN) without a selectable marker was reconstructed. LacZ between fragment C and fragment K in non-replicating recombinant vaccinia virus VTKRG△CKlacZ was deleted and was substituted with rabies virus nucleoprotein by homologous recombination resulting in VTKRGΔCKRN. The recombinant vaccinia virus was screened by a two-step selection under, G418 and with White/Blue plaque color change as well as immunofluorescence. By Dot Blot analysis, RG gene and RN gene were shown to integrate stably into the non-replicating vaccinia recombinants even after 20 passages in CEF cells. The virulence of VTKRGΔCKRN was obviously less than that of VTKRG in naked mouse. The non-replicating characterization of VTKRGΔCKRN was also stable. It was proved that VTKRGΔCKRN could express RG and RN at the same time. VTKRGΔCKRN could elicit viral neutralizing antibody(VNA) similar to that of VTKRG by inoculated into the mice,and thus protect the animals from lethal challenge of rabies virus. It could elicit efficient viral neutralizing antibody(VNA) and protect the dogs from lethal challenge of rabies street strain SBD by intramuscular inoculation at dose of only 1.6×106PFU. This study confirmed the efficacy and safety of non-replicating recombinant vaccinia virus VTKRGΔCKRN.

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