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Virologica Sinica, 18 (5) : 441-445, 2003
Research Article
Construction of Recombinant Vaccinia Virus for Expressing gagpol Gene of Chinese Epidemic HIV-I Strain(CN54,clade B’/C)
National Centerfor AIDS Control and Prevention,Beijing 100050,China
2.National Key Laboratory ofAgricultural Microbiology,Life Science and Technology College,Huazhong Agriculture University,Wuhan 430070,China
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.
  Published online: 5 Oct 2003
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