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．