Transcription unit and use thereof in expression vectors
View Patent ↗A transcription unit constituted by a polynucleotide including the hCMVie virus enhancer, the enhancer having the nucleotide sequence SEQ ID NO: 1, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 1 and essentially having transcription activation properties, and the promoter region of Cyclin-Dependent Kinase 9 (CDK9), the promoter region having the nucleotide sequence SEQ ID NO: 2, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 2 and essentially having a promoter activity.
1. A transcription unit comprising a polynucleotide comprising the following regulatory elements:
a. the hCMVie virus enhancer, said enhancer having the nucleotide sequence SEQ ID NO: 1, or a nucleic acid having at least 70% sequence identity with the sequence SEQ ID NO: 1 and having transcription activation activity,
b. the promoter region of Cyclin-Dependent Kinase 9 (CDK9), said promoter region haying the nucleotide sequence SEQ ID NO: 2, or a nucleic acid having at least 70% sequence identity with the sequence of SEQ ID NO: 2 and having a promoter activity,
c. a nucleotide sequence situated downstream of said promoter region and upstream of the translation initiation site, said nucleotide sequence comprising the 5′ untranslated region (5′ UTR) of the eukaryotic Initiation Factor 4G1 (elF4GI) gene, said 5′ UTR having the nucleotide sequence SEQ ID NO: 5, or a nucleic acid having at least 70% sequence identity with the sequence of SEQ ID NO: 5 and having mRNA stabilization and translation facilitator activitites, and
d. the intron of the Elongation Factor 1 a (EFla) gene having the nucleotide sequence SEQ ID NO: 10, or a nucleic acid having at least 70% sequence identity with the sequence of SEQ ID NO: 10, said intron being situated downstream of said 5′ UTR region and upstream of the transcription initiation site,
said transcription unit having the nucleotide sequence SEQ ID NO: 33, or a nucleotide add haying at least 70% sequence identity with the sequence SEQ ID NO: 33, and having said transcription activation activity, said promoter activity, and said mRNA stabilization and translation facilitator activity,
wherein said transcription unit is capable of generating a volume production of a protein of interest greater than that obtained with a combination of said CMVie virus enhancer combined with said promoter region of CDK9 alone.
2. An expression vector comprising at least one transcription unit as defined according to claim 1 and at least one cloning site allowing the integration of a nucleotide acid coding for a protein of interest.
3. An expression vector comprising at least one transcription unit as defined according to claim 1 and at least one site for the site-specific recombination allowing the integration of a nucleotide acid coding for a protein of interest.
4. The expression vector according to claim 2 , also comprising a eukaryotic resistance gene, a bacterial resistance gene, a bacterial origin of replication and a dedicated gene amplification unit.
5. The expression vector according to claim 2 , in which said protein of interest is chosen from the group constituted by the proteins participating in coagulation or an immunoglobulin, cytokines, hormones, growth factors or complement factors and any fusion protein.
6. A host cell comprising an expression vector as defined in claim 2 .
7. The host cell according to claim 6 , said host cell being the YB2/0 cell line.
8. An expression system comprising an expression vector as defined according to claim 2 and a host cell comprising said expression vector allowing the expression of a protein of interest encoded by a nucleotide acid.