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The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector

1992; Elsevier BV; Volume: 186; Issue: 1 Linguagem: Inglês

10.1016/0042-6822(92)90075-z

1096-0341

Catherine Giraud, G. Devauchelle, Max Bergoin,

CRISPR and Genetic Engineering

An infectious genome of the Junonia coenia densovirus (1c DNV) has been recently cloned and sequenced. We investigated the ability of this cloned genome to be used as expression vector by inserting the lac Z gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence. The resulting recombinant plasmid designated pBRJ/ac Z was transfected into insect SPC-SL 52 cells and the expression of β-galactosidase (β-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates. Western blot analysis revealed that β-gal was expressed as chimeric capsid-β-gal polypeptides. This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence. Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication. Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically. Encapsidation of J/ac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions. Detection of a β-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that J/ac Z recombinant genome could be maintained in an integrative or episomal state.