Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-12-18
1999-08-17
Guzo, David
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4353201, 435455, 435456, 435463, 435466, C12P 2100, C12N 1586, C12N 1563
Patent
active
059392851
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to new expression vectors obtained from baculoviruses.
DESCRIPTION OF THE BACKGROUND
The baculoviruses, represented by the Autographa californica nuclear polyhedrosis virus (AcMNPV), possess several promoters which are active in different phases of the viral replication cycle. Some of these promoters are used in genetic engineering to control the expression of heterologous genes inserted into the baculovirus genome. Among the ones most commonly employed, two strong very late promoters may be mentioned: the polyhedrin (polh) promoter and the P10 polypeptide promoter, which are active only at the end of the infectious cycle, after replication of the viral genome, and which enable the genes placed under their control to be expressed at a high level.
The polh and P10 promoters are described in detail in the following Virol. vol. 70, pp. 1273-1279, (1989)! for the P10 promoter. The sequence localized between positions (-71 and +1) defined relative to the A(+1) of the polyhedrin ATG is generally defined as the "polyhedrin promoter", and the sequence localized between positions (-70 and +1) defined relative to the A(+1) of the p10 polypeptide ATG is generally defined as the "p10 promoter".
The obtaining from a baculovirus, of a vector expressing a foreign gene under transcriptional control of a promoter of the baculovirus, is generally performed by methods known per se, through construction of a transfer vector containing the the promoter, then recombination with the DNA of the wild-type virus, and lastly selection of the recombinants.
SUMMARY OF THE INVENTION
During the replication cycle of the baculovirus, the early genes are expressed first: the transcription of these genes involves the host cell's RNA polymerase II.
Subsequently, the late and very late genes such as polh and P10 are transcribed by a particular RNA polymerase, of at least partially viral origin, which is insensitive to .alpha.-amanitin.
An A/GTAAG motif common to all the late genes constitutes the transcription startsite. This motif is essential for the recognition of these promoters by the RNA polymerase, and hence for their activity. The mechanisms which govern the transcriptional activation are not yet, however, precisely known.
Previous work of the inventors' team has shown that it is especially advantageous, in order to increase the level of expression of a heterologous gene placed under the control of one of the two strong very late promoters polh or P10, to construct a baculovirus in which only one of these two strong late promoters is inactivated, and to cause the said gene to be expressed under the control of the remaining promoter.
This principle has been used as a basis for the construction of several of the INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE (I.N.R.A.) and of the CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (C.N.R.S.); CHAABIHI et al., J. Virol., 67, 2664-2671 (1993)!, in which either the P10 promoter (baculovirus designated AcSLP33, for example) or the polyhedrin promoter (baculovirus designated AcSLP10, for example) is inactivated by deletion.
In the context of continuing their work on the regulation of expression in baculoviruses, it occurred to the inventors to study the influence of the coding sequence of the P10 gene on the expression of the latter. For this purpose, they made, from the wild-type baculovirus AcMNPV, constructs comprising the CAT reporter gene inserted at one of the positions +16, +150 or +230 of the sequence coding for 10 (these positions are defined relative to the A (+1) of the ATG initiation codon of P10), and studied the simultaneous expression of the CAT reporter gene and of the polyhedrin gene in the recombinant viruses obtained (designated AcCAT+16, AcCAT+150 and AcCAT+230, respectively), relative to the wild-type baculovirus AcMNPV and to the modified baculovirus AcSLP33 (in which the P10 promoter is inactivated).
The recombinant viruses AcCAT+16, AcCAT+150 and AcCAT+230 are shown diagrammatically in FIG
Cerutti Martine
Devauchelle Gerard
Ogliastro Marie-Helene
Centre National de la Recherche Scientifique "CNRS"
Guzo David
Institut National de la Recherche Scientifique Agronomique (INRA
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