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Gene Delivery into Mammalian Cells by Recombinant Baculovirus and Antibody Production of NS1from Human Bocavirus

SEYNI YANSAMBOU Mahamadou  
【摘要】:The gene delivery into mammalian cells needs a high titer of the expressed gene. To obtain the high expression of the interested proteins, the baculovirus expression system is usually used to generate the recombinant baculovirus. Phylogenetic analysis shows that the human bocavirus (HBoV) belongs to the genus bocavirus within the subfamily, Parvovirinae; of the family, parvoviridae and is most closely related to the bovine parvovirus (BPV) and the minute virus of canines (MVC). Like these viruses, HBoV1is studied with similar conditions and methods. HBoV1is a newly identified human pathogenic virus in children's nasopharyngeal aspirates from individuals with respiratory tract illnesses. To further study viral characteristics, numerous researches on the nonstructural proteins are directed in this way. This study describes the generation of three recombinant baculoviruses (Bacmid-NS1, Bacmid-NP1and Bacmid-ORFx) under the control of the HBoV gene promoter. Bacmid-NSl and Bacmid-NPl were used to infect insect Sf9(Spodoptera frugiperda) cells to amplify these viruses and then the recombinant baculoviruses were used to transduce mammalian cells (Hela) to detect the expression of these proteins (NS1and NP1). The pFastBac-HTA was used as the vector to clone these protein genes and then transform them into the E.coli DH10Bac competent cells to generate recombinant bacmids. Finally the recombinant bacmids were used to transfect into the insect cells Sf9for the generation of the recombinant virus. These viruses were further expanded to generate a viral stock with a high titer. The recombinant viruses were used to transduce into Hela cells, and the transduced Hela cells were stained using antibodies to detect the recombinant proteins by the Immunofluorescence (IF) method.


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