Highly efficient eukaryotic expression vector comprising an exogenous transcription regulatory element
The present invention relates to a highly efficient eukaryotic expression vector containing an exogenous transcription regulatory element which comprises a promoter/enhancer and the nucleotide sequence upstream of the translation initiation codon derived from human cytomegalovirus (HCMV) immediately early (IE) gene or human elongation factor 1α (EF1α) gene.
1. A transcription regulatory element operably linked to a heterologous coding sequence, the transcription regulatory element consisting of the nucleotide sequence of SEQ ID NO: 3.
2. A eukaryotic expression vector comprising the transcription regulatory element of claim 1 .
3. The vector of claim 2 , further comprising a ColE1 origin of replication in operable association with said transcription regulatory element.
4. The vector of claim 3 , further comprising a poly A signal in operable association with said transcription regulatory element.
5. The vector of claim 2 , further comprising a selectable marker gene in operable association with said transcription regulatory element.
6. The vector of claim 2 , further comprising a multi-cloning site in operable association with said transcription regulatory element.
7. The vector of claim 6 , wherein said transcription regulatory element is located upstream of said multi-cloning site.
8. The vector of claim 7 , further comprising a ColE1 origin of replication in operable association with said transcription regulatory element.
9. The vector of claim 8 , further comprising a poly A signal in operable association with said transcription regulatory element.
10. The vector of claim 9 , wherein said poly A signal is a BGH poly A signal.
11. The vector of claim 10 , further comprising a selectable marker gene in operable association with said transcription regulatory element.
12. The vector of claim 11 , wherein said selectable marker gene is an ampicillin resistance gene.
13. The vector of claim 2 , further comprising:
a multi-cloning site;
a ColE1 origin of replication;
a polyA signal; and
a selectable marker gene;
wherein said multi-cloning site; said ColE1 origin of replication; said polyA signal; and said selectable marker gene are in operable association with said transcription regulatory element.
14. The vector of claim 13 , wherein said selectable marker gene is an ampicillin resistance gene.
15. The vector of claim 13 , wherein said vector is pCN.
16. The vector of claim 13 , wherein said selectable marker gene is a kanamycin resistance gene.
17. The vector of claim 13 , wherein said vector is pCK.
18. The vector of claim 7 , further comprising a nucleotide sequence of interest within said multi-cloning site.
19. The vector of claim 18 , wherein said nucleotide sequence of interest is a cDNA encoding a human VEGF.
20. The vector of claim 19 , wherein said human VEGF is VEGF165.
21. The vector of claim 20 , wherein said vector is pCN/VEGF or pCK/VEGF.
22. A method of delivering a polypeptide into a eukaryotic cell comprising the step of contacting said eukaryotic cell with the vector of any one of claims 18 - 21 .
23. A method of delivering a polypeptide into a mammal comprising the step of administering to the mammal the vector of any one of claims 18 - 21 .
24. An isolated cell transformed with the eukaryotic expression vector of any of claims 1 - 21 , said cell being a microorganism or an animal cell.
25. The cell of claim 24 , wherein the cell is selected from the group consisting of Escherichia coli Top 10-pCN/VEGF (KCCM-10478); Escherichia coli Top 10-pCK (KCCM-10476); and Escherichia coli Top 10-pCK/VEGF 165 (KCCM-10179).
26. A gag-pol expression vector comprising the transcription regulatory element of claim 1 .
27. The gag-pol expression vector of claim 26 , wherein said vector comprises a gag-pol fragment derived from an MLV.
28. The gag-pol expression vector of claim 27 , wherein said vector is pCPu-gag.pol.
29. An env expression vector comprising the transcription regulatory element of claim 1 .
30. The env expression vector of claim 29 , wherein said vector is pCH-env.