National Centerfor, AIDS Control, and Prevention, National Key, Laboratory ofAgricultural, Life Science, and Technology and Huazhong Agriculture. Construction of Recombinant Vaccinia Virus for Expressing gagpol Gene of Chinese Epidemic HIV-I Strain(CN54,clade B’/C)[J]. Virologica Sinica, 2003, 18(5): 441-445.
Citation: National Centerfor, AIDS Control, and Prevention, National Key, Laboratory ofAgricultural, Life Science, and Technology, Huazhong Agriculture. Construction of Recombinant Vaccinia Virus for Expressing gagpol Gene of Chinese Epidemic HIV-I Strain(CN54,clade B’/C) .VIROLOGICA SINICA, 2003, 18(5) : 441-445.

表达HIV-I CN54株gagpol基因重组痘苗病毒疫苗株的构建

  • 摘要:为研制不带筛选标记的HIV活载体疫苗,首先构建含有neo基因和lacZ基因双重筛选标记的pVI75转移 质粒,并将HIV-I中国主要流行株B’/c重组株CN54 gagpol基因置于pVI75的启动子pE/L下,构建重组质粒 pVI75一Gagpol。重组质粒与痘苗病毒天坛株共转染鸡胚细胞。前三轮通过G418加压,噬斑纯化,得到既含目的 基因又含筛选标记的蓝色重组痘苗病毒;后三轮在无G418选择压力下,筛选只含目的基因而缺失了筛选标记的 白色重组痘苗病毒。结果表明筛选到了一株重组病毒,经PCR和Dot blot检测确认该株重组痘苗病毒的?180基因 和lacZ基因已丢失:PCR鉴定表明目的基因已插入重组痘苗病毒中;抗体染色和Westernblot结果证实该重组病 毒能很好地表达目的蛋白。

Construction of Recombinant Vaccinia Virus for Expressing gagpol Gene of Chinese Epidemic HIV-I Strain(CN54,clade B’/C)

  • Abstract:To develop live—vectored vaccine of Human immunodeficiency virus (HIV-I)without selectable marker,we first constructed a tran sfer plasmid pVI75 with selectable m arkers of neo an d/acZ gene,an d a recombinant plasmid pVI75一Gagpol containing the gagpol gene of Chinese predominan t prevalent HIV-I strain CN54,pE/L upstream as the promoter.CEF was tran sfected by the recombinan t plasmid pVI75一Gagpol,one hour after being infected with Tiantan vaccinia virus.A recombinan t vaccinia virus rVV-Gagpol without selectable marker was constructed through two homologous recombinations as following:first,through three cycles of plaque purification under G4 1 8 pressure,the blue recombinan t virus including both ga~ ol gene and the selectable marker gene was acquired;then through three cycles of plaque purification without G4 1 8 pressure,the white recombinan t virus with ga~ ol gene but without the selectable marker gene was acquired.Thus,a recombinan t vaccinia virus was acquired.PCR an d Dot blot assay showed that the recombinant rVV-Gagpol lost the neo gene and lacZ gene.Gagpol gene could be detected by PCR.Antibody staining an d Western blot results indicated this recombinant vaccinia virus could successfully express HIV Gagpol protein.

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    Construction of Recombinant Vaccinia Virus for Expressing gagpol Gene of Chinese Epidemic HIV-I Strain(CN54,clade B’/C)

    • 1. National Centerfor AIDS Control and Prevention,Beijing 100050,China

    Abstract: Abstract:To develop live—vectored vaccine of Human immunodeficiency virus (HIV-I)without selectable marker,we first constructed a tran sfer plasmid pVI75 with selectable m arkers of neo an d/acZ gene,an d a recombinant plasmid pVI75一Gagpol containing the gagpol gene of Chinese predominan t prevalent HIV-I strain CN54,pE/L upstream as the promoter.CEF was tran sfected by the recombinan t plasmid pVI75一Gagpol,one hour after being infected with Tiantan vaccinia virus.A recombinan t vaccinia virus rVV-Gagpol without selectable marker was constructed through two homologous recombinations as following:first,through three cycles of plaque purification under G4 1 8 pressure,the blue recombinan t virus including both ga~ ol gene and the selectable marker gene was acquired;then through three cycles of plaque purification without G4 1 8 pressure,the white recombinan t virus with ga~ ol gene but without the selectable marker gene was acquired.Thus,a recombinan t vaccinia virus was acquired.PCR an d Dot blot assay showed that the recombinant rVV-Gagpol lost the neo gene and lacZ gene.Gagpol gene could be detected by PCR.Antibody staining an d Western blot results indicated this recombinant vaccinia virus could successfully express HIV Gagpol protein.

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