Modified baculovirus and baculovirus expression vectors

Details Modified baculovirus and baculovirus expression vectors Modified baculovirus and baculovirus expression vectors

1993-06-01

1996-11-05

Guzo, David

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

4353201, 4351721, 4351723, 435 691, C12N 1586, C12N 700

Patent

active

055717098

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to expression vectors obtained from modified baculoviruses, in which one of the two strong late promoters of the wild-type baculovirus is inactive.


BACKGROUND OF THE INVENTION

Baculoviruses are currently used in a very large number of laboratories as vectors for the expression of genes. In effect, these viruses possess the following advantages: they permit the insertion of long segments of DNA and possess, in addition, two strong late promoters, the polyhedrin promoter and the P10 protein promoter, which are capable of inducing an extremely high level of expression of the genes placed under their control.
The publication of L. K. MILLER [CHAPTER 14 "A VIRUS VECTOR FOR GENETIC ENGINEERING IN INVERTEBRATES" of the manual "GENETIC ENGINEERING IN THE PLANT SCIENCES" (1981) N. J. PANAPOULOS, ed., Praeger Pub. New York, p. 203-224] describes the advantages of the use of baculoviruses, and especially of the baculovirus Autographa californica (AcNPV), as vectors for the expression of foreign genes in insect host cells. The AcNPV baculovirus described in this publication possesses two mature forms, referred to as the non-occlusive form (NOV) and the occlusive form (OV), respectively. The non-occlusive forms are responsible for transmission of the virus in cell culture or within the same organism. The occlusive forms are responsible for transmission of the virus from one organism to another. These occlusive forms consist of virions sheathed in a crystalline protein matrix consisting essentially of a single protein: polyhedrin.
It was demonstrated that the polyhedrin gene possessed an exceptionally strong promoter which enabled a high level of expression of the gene placed under its control to be effected. MILLER hence points out that it would be especially advantageous to use baculoviruses as expression vectors, inserting under the control of the polyhedrin promoter the exogenous DNA which it is desired to express.
Other work [SMITH and SUMMERS, J. Virol. 45, 215-225 (1983)] showed that the AcNPV virus possessed another strong late promoter, which is the promoter of the 10 kDa polypeptide or P10 protein.
In addition, the capsid of baculoviruses can contain large amounts of DNA, and hence does not constitute a limit to the number of genes inserted or to their length. Consequently, a very large number of expression vectors derived from baculoviruses have been proposed. For example, European Patent Application 127,839 (SUMMERS invention) describes a method enabling a recombinant expression vector derived from baculovirus to be produced, proceeding via a transfer vector containing a viral DNA fragment containing the polyhedrin gene promoter, downstream of which the foreign gene is inserted. This transfer vector is then recombined with wild-type baculovirus DNA, and the recombinants possessing the foreign gene under the control of the polyhedrin promoter are selected.
European Patent Application 340,359 (inventors PAGE and ROGERS) describes transfer vectors derived from baculoviruses, in which the polyhedrin ATG initiation codon is eliminated, so that the sequence coding for polyhedrin cannot be translated, and which carries a restriction site suitable for insertion of an exogenous gene downstream of the N-terminal end of the polyhedrin gene, and in immediate proximity to the said end.
The Inventors' team has previously developed a method for producing a modified baculovirus which is usable as an expression vector, in which a suitable restriction site is inserted directly, and without recourse to a transfer vector, downstream of a strong late promoter. The vector thereby obtained may be loaded, at the position of the restriction site introduced, with the gene which it is desired to have expressed; this method forms the subject of European Patent Application 345,152.
In all the known methods, the gene to be expressed is inserted under the control of one of the two strong late promoters present in the genome of the wild-type baculovirus, while the other promoter continues to function normal

REFERENCES:
Qin et al. "Studies on the Control Region of the p/o Gene of the Autographs calfornica Nuclear Polyhedrosis Virus" J. Gen. Virol., vol. 70, pp. 1273-1279, 1989.
Weyer et al. "Analysis of the Promoter of the Autographs cablfrnica Nuclear Polyhedrosis Virus p. 10 Gene", J. Gen. Virol., vol. 70, pp. 203-208, 1989.
Vatrou et al. "Bombyx morie Nuclear Polyhedrous virus-based vectors for expressing pasenger genes in silkworm cells under viral or cellalar promoter control", Gens, vol. 75, pp. 59-71, 1989.
Hassan Chaabifi, "Investigation of the Regulation of p. 10 and polyhedrin genes of the Autographs californica baculoviues and development of new vectors for expressing foreign genes", Ph.D. Theais Univeisite I'Aix-Marseille 2, France, 1992, Abstact.
van Oers et al. "Expression of the Autographs californica nuclear polyhedrosis virus p. 10 gene: effect of polyhdedrin gene expression", Arch. Virol. (1992), vol. 123, pp. 1-11.

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