Baculovirus expression system
The present disclosure relates to a heterologous recombinant baculovirus (rBV) expression system for the production of foreign heterologous proteins in insect cells. This system comprises a recombinant baculovirus backbone within a genome with a deletion in the cathepsin gene into which foreign gene cassettes can be integrated, and an insect cell that can be infected by the Δv-cath-rBV, and in which the foreign proteins and/or viral vectors or particles are expressed.
1 . A recombinant baculovirus (rBV) genome comprising an rBV DNA backbone, wherein the rBV DNA backbone comprises:
(i) a chitinase gene;
(ii) a deletion of a v-cath gene, and
(iii) a DNA fragment enabling integration of one or more protein expression cassettes into the rBV DNA backbone, wherein the one or more protein expression cassettes comprise:
at least one gene comprising a sequence encoding an AAV capsid protein,
an insect cell promoter operably linked to the at least one gene, and
two DNA sequences enabling the one or more protein expression cassettes to integrate into the rBV DNA backbone; and
upon infection of the rBV genome into an insect cell, AAV vectors are produced at a level about 3 times greater as compared to AAV vectors produced from an rBV DNA backbone without a deletion of the v-cath gene.
2 . The rBV genome of claim 1 , wherein the DNA fragment of the rBV DNA backbone comprises a DNA sequence homologous to two sequences flanking the one or more protein expression cassettes in a donor plasmid.
3 . The rBV genome of claim 1 , wherein the DNA fragment of the rBV DNA backbone is derived from bMON14272.
4 . The rBV genome of claim 3 , wherein the DNA fragment of the rBV DNA backbone comprises an origin of replication.
5 . The rBV genome of claim 3 , wherein the DNA fragment of the rBV DNA backbone further comprises a reporter gene.
6 . The rBV genome of claim 1 , wherein the rBV DNA backbone further comprises a selection marker expression gene cassette integrated into the v-cath deletion.
7 . The rBV genome of claim 1 , wherein the two DNA sequences enabling the one or more protein expression cassettes to integrate into the rBV DNA backbone are homologous to the DNA sequences in the rBV DNA backbone or are transposable elements.
8 . A recombinant baculovirus (rBV) vector or particle comprising:
the rBV genome of claim 1 ; and
at least one baculoviral capsid protein.
9 . An isolated insect cell comprising the rBV vector or particle of claim 8 .
10 . The insect cell of claim 9 , further comprising at least one AAV capsid protein expressed from the one or more protein expression cassettes in the rBV DNA backbone of the rBV genome.
11 . A heterologous expression system comprising:
the rBV vector or particle of claim 8 ; and
an insect cell susceptible to infection and capable of expressing at least one AAV capsid protein encoded by the rBV DNA backbone in the rBV vector or particle.
12 . A non-viscous insect cell lysate, comprising:
the rBV genome of claim 1 ; and
at least one AAV capsid protein encoded by the rBV DNA backbone and expressed in the lysate.
13 . The rBV genome of claim 1 , wherein the DNA fragment enabling integration of one or more protein expression cassettes into the rBV DNA backbone comprises a transposon fragment.
14 . The rBV genome of claim 1 , wherein the one or more protein expression cassettes comprise sequences encoding AAV capsid proteins VP1, VP2, and VP3, and upon infection of the rBV genome into an insect cell, AAV vectors are produced at a level about 4 times greater as compared to AAV vectors produced from an rBV DNA backbone without a deletion of the v-cath gene.