# US 5,348,886 A · Method of producing recombinant eukaryotic viruses in bacteria > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,348,886 A - Document ID: US5348886A - Status: Granted - Kind: A - Application number: 07/941,363 - Filed: 1992-09-04 - Granted: 1994-09-20 - Earliest claimed: 1992-09-04 - Assignees: Monsanto Company - Inventors: Stephen C. Lee; Mark S. Leusch; Verne A. Luckow; Peter O. Olins - Web page: https://patentgrep.com/patent/US5348886A ## CPC - C12N 15/86: Viral vectors - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C12N 2710/14143: viral genome or elements thereof as genetic vector - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A method for producing infectious recombinant baculoviruses in bacteria is described. A novel baculovirus shuttle vector (bacmid) was constructed that contains a low-copy-number bacterial replicon, a selectable drug resistance marker, and a preferred attachment site for a site-specific bacterial transposon, inserted into a nonessential locus of the baculovirus genome. This shuttle vector can replicate in E. coli as a plasmid and is stably inherited and structurally stable after many generations of growth. Bacmid DNA isolated from E. coli is infectious when introduced into susceptible lepidopteran insect cells. DNA segments containing a viral promoter driving expression of a foreign gene in insect cells that are flanked by the left and right ends of the site-specific transposon can transpose to the attachment site in the bacmid propagated in E. coli when transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells. ## Claims ### Claim 1 A Bacmid, comprising; a. a nuclear polyhedrosis virus DNA which includes the elements required for said nuclear polyhedrosis virus DNA propagation in insect cells; b. a low copy number bacterial replicon, inserted into a nonessential locus of said nuclear polyhedrosis virus DNA, which drives the replication of said nuclear polyhedrosis virus DNA in bacteria; c. a bacterial genetic marker inserted into nonessential locus of said nuclear polyhedrosis virus DNA; and d. a preferential target site for the insertion of a transposon inserted into a nonessential locus of said nuclear polyhedrosis virus DNA. ### Claim 2 (depends on 1) A Bacmid as recited in claim 1 wherein said nuclear polyhedrosis virus DNA is a member of the Multiple nucleocapsids per envelope (MNPV) subgenera of the Nuclear polyhedrosis virus (NPV) genera of the Eubaculovirinae subfamliy (occluded baculoviruses) of the Baculoviridae family of insect viruses. ### Claim 3 (depends on 1) A Bacmid as recited in claim 1 wherein said bacterial replicon is mini-F. ### Claim 4 (depends on 1) A Bacmid as recited in claim 1 wherein said bacterial genetic marker is a selectable marker. ### Claim 5 (depends on 1) A Bacmid as recited in claim 1 wherein said preferential target site is attTn7. ### Claim 6 (depends on 1) A Bacmid as recited in claim 1 wherein said bacterium is E. coli. ### Claim 7 (depends on 1) The bacmid as recited in claim 1 wherein said bacmid is selected from the group consisting of A.T.C.C. 69059 and 69060. ### Claim 8 (depends on 2) A Bacmid as recited in claim 2 wherein said nuclear polyhedrosis virus DNA is the Autographa californica nuclear polyhedrosis virus species of the Multiple nucleocapsids per envelope (MNPV) subgenera of the Nuclear polyhedrosis virus (NPV) genera of the Eubaculovirinae subfamily (occluded baculoviruses) of the Baculoviridae family of insect viruses. ### Claim 9 (depends on 4) A Bacmid as recited in claim 4 wherein said selectable marker confers ampicillin, tetracycline, kanamycin, or gentamicin resistance. ### Claim 10 (depends on 9) A Bacmid as recited in claim 9 wherein said selectable marker confers kanamycin resistance. ### Claim 11 A donor DNA molecule, comprising: a. A bacterial replicon; and b. A transposon operably linked to said bacterial replicon that can be transposed site-specifically into a preferential target site and which includes a heterologous DNA and a bacterial genetic marker. ### Claim 12 (depends on 11) The Donor DNA molecule of claim 11 wherein said DNA molecule is a Donor plasmid. ### Claim 13 (depends on 11) The donor DNA molecule of claim 11 wherein said bacterial replicon is the bacterial chromosome. ### Claim 14 (depends on 11) The donor DNA molecule of claim 11 wherein said heterologous DNA is under the control of a promoter which is operable in Eukaryotic host cells. ### Claim 15 (depends on 12) The donor plasmid of claim 12 wherein said transposon is Tn7. ### Claim 16 (depends on 12) The donor plasmid of claim 12 wherein said bacterial replicon is temperature-sensitive. ### Claim 17 (depends on 12) The donor plasmid of claim 12 wherein said bacterial replicon is incompatible with the helper plasmid. ### Claim 18 (depends on 12) The donor plasmid as recited in claim 12 wherein said heterologous DNA is under the control of a promoter which is operable in insect cells. ### Claim 19 (depends on 12) The donor plasmid as recited in claim 12 wherein said donor plasmid is selected from the group consisting of A.T.C.C. 69061 and 69062. ### Claim 20 A composite Bacmid, comprising: a. a nuclear polyhedrosis virus DNA which includes the elements required for said nuclear polyhedrosis virus DNA propagation in insect cells; b. a low copy number bacterial replicon, inserted into a nonessential locus of said nuclear polyhedrosis virus DNA, which drives the replication of said nuclear polyhedrosis virus DNA in bacteria; c. a first bacterial genetic marker inserted into a nonessential locus of said nuclear polyhedrosis virus DNA; d. a preferential target site for the insertion of a transposon inserted into a nonessential locus of said nuclear polyhedrosis virus DNA; and e. a transposon, inserted into said preferential target site, which includes heterologous DNA and a second bacterial genetic marker that is different from said first bacterial genetic marker. ### Claim 21 (depends on 20) A composite Bacmid as recited in claim 20 wherein said nuclear polyhedrosis virus DNA is a member of the Multiple nucleocapsids per envelope (MNPV) subgenera of the Nuclear polyhedrosis virus (NPV) genera of the Eubaculovirinae subfamily (occluded baculoviruses) of the Baculoviridae family of insect viruses. ### Claim 22 (depends on 20) A composite bacmid as recited in claim 20 wherein said heterologous DNA is under the control of a promoter that is operable in insect cells. ### Claim 23 (depends on 20) A composite bacmid as recited in claim 20 wherein said bacterial replicon is mini-F. ### Claim 24 (depends on 20) A composite bacmid as recited in claim 20 wherein said bacterial genetic marker is a selectable marker. ### Claim 25 (depends on 20) A composite bacmid as recited in claim 20 wherein said preferential target site is attTn7. ### Claim 26 (depends on 20) A composite bacmid as recited in claim 20 wherein said bacteria are E. coli. ### Claim 27 (depends on 21) A composite Bacmid as recited in claim 21 wherein said nuclear polyhedrosis virus DNA is the Autographa californica nuclear polyhedrosis virus species of the Multiple nucleocapsids per envelope (MNPV) subgenera of the Nuclear polyhedrosis virus (NPV) genera of the Eubaculovirinae subfamily (occluded baculoviruses) of the Baculoviridae family of insect viruses. ### Claim 28 (depends on 24) A composite bacmid as recited in claim 24 wherein said selectable marker confers ampicillin, tetracycline, kanamycin or gentamicin resistance. ### Claim 29 (depends on 24) A composite bacmid as recited in claim 24 wherein said selectable marker confers kanamycin resistance. ### Claim 30 (depends on 25) A composite bacmid as recited in claim 25 wherein said attTn7 is inserted into the middle of the lacZ.alpha. region. ### Claim 31 A method for producing a composite bacmid, which comprises; A. introducing into bacteria the following components in any order or all together; 1. a Bacmid, comprising; a. a nuclear polyhedrosis virus DNA which includes the elements required for said nuclear polyhedrosis virus DNA propagation in insect cells; b. a low copy number bacterial replicon, inserted into a nonessential locus of said nuclear polyhedrosis virus DNA, which drives the replication of said nuclear polyhedrosis virus DNA in bacteria; c. a bacterial genetic marker inserted into a nonessential locus of said nuclear polyhedrosis virus DNA; and d. a preferential target site for the insertion of a transposon inserted into a nonessential locus of said nuclear polyhedrosis virus DNA; 2. a donor DNA molecule, comprising; a. a bacterial replicon; and b. a transposon operably linked to said bacterial replicon that can be transposed site-specifically into a preferential target site on a bacmid and which includes a heterologous DNA and a bacterial genetic marker. 3. a helper plasmid, wherein components 1, 2 and 3 have different bacterial genetic markers; B. Incubating said bacteria; C. Identifying bacteria in which transposition has occurred; and D. Isolating a composite bacmid from said identified bacteria. ### Claim 32 (depends on 31) The method for producing a composite Bacmid as recited in claim 31 wherein said nuclear polyhedrosis virus DNA is a member of the Multiple nucleocapsids per envelope (MNPV) subgenera of the Nuclear polyhedrosis virus (NPV) genera of the Eubaculovirinae subfamily (occluded baculoviruses) of the Baculoviridae family of insect viruses. ### Claim 33 (depends on 31) The method for producing a composite Bacmid as recited in claim 31 wherein said bacterial replicon is mini-F. ### Claim 34 (depends on 31) The method for producing a composite Bacmid as recited in claim 31 wherein said bacterial genetic marker is a selectable marker. ### Claim 35 (depends on 31) The method for producing a composite Bacmid as recited in claim 31 wherein said preferential target site is attTn7. ### Claim 36 (depends on 31) The method for producing a composite Bacmid as recited in claim 31 wherein said bacteria are E. coli. ### Claim 37 (depends on 31) The method for producing a composite Bacmid as recited in claim 31 wherein said nuclear polyhedrosis virus DNA is under the control of a promotor capable of driving the expression of heterologous protein in insect cells. ### Claim 38 (depends on 32) The method for producing a composite Bacmid as recited in claim 32 wherein said nuclear polyhedrosis virus DNA is the Autographa californica nuclear polyhedrosis virus species of the Multiple nucleocapsids per envelope (MNPV) subgenera of the Nuclear polyhedrosis virus (NPV) genera of the Eubaculovirinae subfamily (occluded baculoviruses) of the Baculoviridae family of insect viruses. ### Claim 39 (depends on 34) The method for producing a composite Bacmid as recited in claim 34 wherein said selectable marker confers ampicillin, tetracycline, kanamycin, or gentamicin resistance. ### Claim 40 (depends on 34) The method for producing a composite Bacmid as recited in claim 34 wherein said selectable marker confers kanamycin resistance. ### Claim 41 (depends on 35) The method for producing a composite Bacmid as recited in claim 35 wherein said attTn7 is inserted into the middle of the lacZ.alpha. region. ### Claim 42 (depends on 20) A method of producing heterologous protein in insect cells using a composite bacmid of claim 20 comprising; 1. Introducing into said insect cells said composite Bacmid; 2. Incubating said insect cells; and 3. Isolating said heterologous protein.