CA2558864A1

CA2558864A1 - Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome

Info

Publication number: CA2558864A1
Application number: CA002558864A
Authority: CA
Inventors: Friedrich Dorner; Friedrich Scheiflinger; Falko Gunter Falkner; Michael Pfleiderer
Original assignee: Immuno Aktiengesellschaft; Friedrich Dorner; Friedrich Scheiflinger; Falko Gunter Falkner; Michael Pfleiderer; Baxter Aktiengesellschaft; Baxter Healthcare S.A.
Current assignee: Baxter Healthcare SA
Priority date: 1991-08-26
Filing date: 1992-08-25
Publication date: 1993-02-27
Anticipated expiration: 2012-08-25
Also published as: CA2558864C

Abstract

A method is disclosed for producing a modified eukaryotic cytoplasmic DNA virus by direct molecular cloning of a modified DNA molecule comprising a modified cytoplasmic DNA virus genome. The inventive method comprises the steps of (I) modifying under extracellular conditions a DNA molecule comprising a first cytoplasmic DNA virus genome to produce a modified DNA molecule comprising the modified cytoplasmic DNA virus genome; (II) introducing the modified DNA molecule into a first host cell which packages the modified DNA molecule into infectious virions; and (III) recovering from the host cell virions comprised of the modified viral genome. The host cell is infected with a helper virus which is expressed to package the modified viral genome into infectious virions. Examples of packaging a modified poxvirus genome by a helper poxvirus of the same or different genus are described. Also disclosed are novel poxvirus vectors for direct molecular cloning of open reading frames into a restriction enzyme cleavage site that is unique in the vector. In one model poxvirus vector, the open reading frame is transcribed by a promoter located in the vector DNA upstream of a multiple cloning site comprised of several unique cleavage sites.

Claims

1. A plasmid comprising a DNA segment having a cleavage site for the bacterial restriction endonuclease NotI at each end, wherein said DNA segment comprises a sequence-specific endonuclease cleavage site that is unique in said plasmid.

2. A plasmid comprising a DNA segment having a cleavage site for the bacterial restriction endonuclease NotI at each end, wherein said DNA segment comprises a sequence-specific endonuclease cleavage site that is unique in said plasmid, and wherein said plasmid is as shown in Figure 1.3, designated pN2 and comprising the sequences of SEQ. ID. NO. 1.

3. The plasmid according to Claim 1, wherein said DNA segment further comprises a selective marker gene under transcriptional control of a chordopoxvirus promoter.

4. The plasmid according to Claim 3, as shown in Figure 1.3, selected from the group of plasmids designated pN2-gpta comprising the sequence of SEQ. ID. NO. 2, and pN2-gptb comprising the sequence of SEQ. ID. NO. 3.

5. The plasmid according to Claim 3, wherein said DNA segment further comprises a second chordopoxvirus promoter operatively linked to a DNA sequence comprising a restriction endonuclease cleavage site.

6. The plasmid according to Claim 5, as shown in Figure 4.7, designated pN2gpt-S4 and comprising the sequence of SEQ. ID. NO. 14.

7. The plasmid according to Claim 5, further comprising a translation start codon operatively linked to said DNA sequence comprising a restriction endonuclease cleavage site.

8. The plasmid according to Claim 7, as shown in Figure 4.7, designated pN2gpt-S3A and comprising the sequence of SEQ. ID. NO. 13.

9. A plasmid comprising a first master cloning site that includes unique sites of a second master cloning site of the vaccinia virus vector designated vdTK, selected from the following group of plasmids shown in Figure 4.3: pA0 comprising the sequence of SEQ. ID. NO. 6, pA1 comprising the sequence of SEQ. ID. NO. 7, and pA2 comprising the sequence of SEQ. ID. NO. 8.

10. The plasmid according to Claim 9, further comprising a chordopoxvirus promoter and a translation start codon operatively linked to said first master cloning site, as shown in Figure 4.4, selected from the group of plasmids:
pA1-S1 comprising the sequence of SEQ. ID. NO. 9 and pA2-S1 comprising the sequence of SEQ. ID. NO. 10.

11. The plasmid according to Claim 9, further comprising a chordopoxvirus promoter operatively linked to said first master cloning site, as shown in Figure 4.5, selected from the group of plasmids: pA1-S2 comprising the sequence of SEQ. ID. NO. 11, and pA2-S2 comprising the sequence of SEQ. ID. NO. 12.

12. The plasmid according to Claim 10, wherein said plasmid further comprises a DNA sequence encoding human prothrobin, wherein further said DNA sequence is operatively linked to said chordopoxvirus promoter and said start codon.

13. The plasmid according to Claim 10, as shown in Figure 5.1, designated plasmid pA1S1-PT, and comprising the sequence of SEQ. ID. NO. 15.

14. The plasmid according to Claim 3, wherein said DNA segment further comprises a second chordopoxvirus promoter operatively linked to a DNA sequence encoding human plasminogen.

15. The plasmid according to Claim 14, selected from the group of plasmids: pN2gpt-GPg, as shown in Figure 5.2, encoding human glu-plasminogen and comprising the sequence of SEQ. ID. NO. 17, and pN2gpt-LPg, as shown in Figure 5.3, encoding lys-plasminogen and comprising a sequence of SEQ. ID. NO. 18.

16. The plasmid according to Claim 3, wherein said DNA segment further comprises a second chordopoxvirus promoter operatively linked to a DNA sequence encoding human immunodeficiency virus (HIV) gp160.

17. The plasmid according to Claim 16, as shown in Figure 5.4, designated pN2gpt-gp160 and comprising the sequence of SEQ. ID. NO. 19.

18. A plasmid comprising a modified EcoRI K fragment of vaccinia virus DNA from which a K1L host range gene is deleted.

19. The plasmid according to Claim 18, as shown in Figure 8.1, selected from the group of plasmids: pEcoK-dhr comprising the sequence of SEQ. ID. NO. 21, and pdhr-gpt comprising the sequence of SEQ. ID. NO. 22.

20. A plasmid comprising a segment of a chordopoxvirus genome that comprises a thymidine kinase gene of said chordopoxvirus, wherein said thymidine kinase gene has been modified to prevent expression of active thymidine kinase, wherein said plasmid is as shown in Figure 4.2 and is selected from the group of plasmids: pHindJ-2 comprising the sequence of SEQ. ID. NO. 4, and pHindJ-3 comprising the sequence of SEQ. ID. NO. 5.

21. The plasmid according to Claim 16, as shown in Figure 9.1, designated jpP2-gp160MN and comprising the sequence of SEQ. ID. NO. 69.

22. The plasmid according to Claim 3, wherein said DNA segment further comprises a second chordopoxvirus promoter operatively linked to a DNA sequence encoding human protein S.

23. The plasmid according to Claim 22, designated pN2-gptaProtS, as shown in Figure 10.1, encoding human protein S and comprising the sequence SEQ. ID. NO 67.

24. The plasmid according to Claim 3, wherein said DNA segment further comprises a second chordopoxvirus promoter operatively linked to a DNA sequence encoding human factor IX.

25. The plasmid according to Claim 24, designated pN2-gpta-FIX, as shown in Figure 11.1, encoding human factor IX and comprising the sequence of SEQ. ID. NO. 72.

26. The plasmid of any one of Claims 1 to 25 for use in a method for producing a modified chordopoxvirus, wherein said method comprises the steps of:
(I) modifying under extracellular conditions a first chordopoxvirus genome to produce a modified chordopoxvirus genome;

(II) infecting a cultured host cell with a second chordopoxvirus that is heterologous to said first chordopoxvirus, said second chordopoxvirus comprising a second viral genome which is expressed in said cultured host cell to package said modified chordopoxvirus genome into an infectious virion;
(III) introducing said modified chordopoxvirus genome into said cultured host cell, wherein said modified chordopoxvirus genome is packaged into an infectious virion;
and (IV) recovering from said cultured host cell said infectious virion that includes said modified chordopoxvirus genome.