CA2558864A1 - Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome - Google Patents
Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome Download PDFInfo
- Publication number
- CA2558864A1 CA2558864A1 CA002558864A CA2558864A CA2558864A1 CA 2558864 A1 CA2558864 A1 CA 2558864A1 CA 002558864 A CA002558864 A CA 002558864A CA 2558864 A CA2558864 A CA 2558864A CA 2558864 A1 CA2558864 A1 CA 2558864A1
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- Canada
- Prior art keywords
- sequence
- seq
- chordopoxvirus
- plasmid
- modified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000700605 Viruses Species 0.000 title abstract 6
- 230000001086 cytosolic effect Effects 0.000 title abstract 5
- 238000010369 molecular cloning Methods 0.000 title abstract 3
- 108020004414 DNA Proteins 0.000 claims abstract 21
- 238000003776 cleavage reaction Methods 0.000 claims abstract 8
- 230000007017 scission Effects 0.000 claims abstract 8
- 210000002845 virion Anatomy 0.000 claims abstract 6
- 238000010367 cloning Methods 0.000 claims abstract 5
- 208000015181 infectious disease Diseases 0.000 claims abstract 5
- 230000002458 infectious effect Effects 0.000 claims abstract 5
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract 5
- 239000013598 vector Substances 0.000 claims abstract 5
- 238000000034 method Methods 0.000 claims abstract 3
- 230000003612 virological effect Effects 0.000 claims abstract 3
- 239000013612 plasmid Substances 0.000 claims 39
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 8
- 108091081024 Start codon Proteins 0.000 claims 3
- 108020004440 Thymidine kinase Proteins 0.000 claims 3
- 108010042407 Endonucleases Proteins 0.000 claims 2
- 102000004533 Endonucleases Human genes 0.000 claims 2
- 101000577630 Homo sapiens Vitamin K-dependent protein S Proteins 0.000 claims 2
- 241000725303 Human immunodeficiency virus Species 0.000 claims 2
- 241000700618 Vaccinia virus Species 0.000 claims 2
- 230000001580 bacterial effect Effects 0.000 claims 2
- 102000052932 human PROS1 Human genes 0.000 claims 2
- 229960000027 human factor ix Drugs 0.000 claims 2
- 229940099815 human protein s Drugs 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 claims 2
- 101000605403 Homo sapiens Plasminogen Proteins 0.000 claims 1
- 108010051456 Plasminogen Proteins 0.000 claims 1
- 102000013566 Plasminogen Human genes 0.000 claims 1
- 102000006601 Thymidine Kinase Human genes 0.000 claims 1
- 239000012634 fragment Substances 0.000 claims 1
- 108010087750 lysyl-plasminogen Proteins 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000003550 marker Substances 0.000 claims 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 claims 1
- 102000053602 DNA Human genes 0.000 abstract 5
- 108700026244 Open Reading Frames Proteins 0.000 abstract 2
- 238000004806 packaging method and process Methods 0.000 abstract 1
- 238000011144 upstream manufacturing Methods 0.000 abstract 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 (26)
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.
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.
(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.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2617830A CA2617830C (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
| CA002616717A CA2616717A1 (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
| CA002616698A CA2616698C (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/750,080 | 1991-08-26 | ||
| US07/750,080 US5445953A (en) | 1991-08-26 | 1991-08-26 | Direct molecular cloning of a modified poxvirus genome |
| US91473892A | 1992-07-20 | 1992-07-20 | |
| US07/914,738 | 1992-07-20 | ||
| CA002515166A CA2515166C (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002515166A Division CA2515166C (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
Related Child Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002616698A Division CA2616698C (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
| CA2617830A Division CA2617830C (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
| CA002616717A Division CA2616717A1 (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2558864A1 true CA2558864A1 (en) | 1993-02-27 |
| CA2558864C CA2558864C (en) | 2009-12-08 |
Family
ID=37080999
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002558864A Expired - Fee Related CA2558864C (en) | 1991-08-26 | 1992-08-25 | Direct molecular cloning of a modified eukaryotic cytoplasmic dna virus genome |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2558864C (en) |
-
1992
- 1992-08-25 CA CA002558864A patent/CA2558864C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2558864C (en) | 2009-12-08 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |