Transcription units and the use thereof in expression vectors
View Patent ↗Novel transcription units that may be used in expression vectors. The transcription unit allow antibodies to be produced whose gain in productivity is not linked to a particular antigenic target antibody and therefore by extrapolation to a given recombinant protein, nor linked to the culture medium.
1. A transcription unit comprising a polynucleotide comprising a sequence selected from the group consisting of:
(a) the sequence SEQ ID NO: 21, comprising:
(i) a hCMVie virus enhancer consisting of nucleotide sequence SEQ ID NO: 1 and possessing an activity of transcription activation,
(ii) a promoter region of B-actin consisting of nucleotide sequence SEQ ID NO: 3 and possessing a promoter activity, and
(iii) a regulatory region R of 5′ Long Terminal Repeat (LTR) of the HTLV-1 virus consisting of nucleotide sequence SEQ ID NO: 4 and having properties of stabilization of mRNA and a translation facilitator;
(b) the sequence SEQ ID NO: 22, comprising:
(i) a hCMVie virus enhancer consisting of nucleotide sequence SEQ ID NO: 1 and possessing an activity of transcription activation,
(ii) a promoter region of R-actin consisting of nucleotide sequence SEQ ID NO: 3 and possessing a promoter activity, and
(iii) a 5′ Untranslated Region (UTR) of a NF-KB Repressing Factor (NRF) gene consisting of nucleotide sequence SEQ ID NO: 5 and having properties of stabilization of mRNA and a translation facilitator;
(c) the sequence SEQ ID NO: 23, comprising:
(i) a hCMVie virus enhancer consisting of nucleotide sequence SEQ ID NO: 1 and possessing an activity of transcription activation,
(ii) a promoter region of R-actin consisting of the nucleotide sequence SEQ ID NO: 3 and possessing a promoter activity, and
(iii) a 5′ UTR region of an eukaryotic Initiation Factor 4GI (eIF4GI) gene consisting of nucleotide sequence SEQ ID NO: 6 and having properties of stabilization of mRNA and a translation facilitator;
(d) the sequence SEQ ID NO: 24, comprising:
(i) a hCMVie virus enhancer consisting of nucleotide sequence SEQ ID NO: 1 and possessing an activity of transcription activation,
(ii) a promoter region of R-actin consisting of the nucleotide sequence SEQ ID NO: 3 and possessing a promoter activity, and
(iii) a 5′ UTR region consisting of sequence SEQ ID NO: 7 and having properties of stabilization of mRNA and a translation facilitator;
(e) the sequence SEQ ID NO: 25, comprising:
(i) a hCMVie virus enhancer consisting of nucleotide sequence SEQ ID NO: 1 and possessing an activity of transcription activation,
(ii) a promoter region of B-actin consisting of nucleotide sequence SEQ ID NO: 3 and possessing a promoter activity, and
(iii) a 5′ UTR region consisting of sequence SEQ ID NO: 8 and having properties of stabilization of mRNA and a translation facilitator;
(f) the sequence SEQ ID NO: 26, comprising:
(i) a hCMVie virus enhancer consisting of nucleotide sequence SEQ ID NO: 1 and possessing an activity of transcription activation,
(ii) a promoter region of B-actin consisting of nucleotide sequence SEQ ID NO: 3 and possessing a promoter activity, and
(iii) a 5′ UTR region consisting of sequence SEQ ID NO: 9 and having properties of stabilization of mRNA and a translation facilitator; and
(g) the sequence SEQ ID NO: 27, comprising:
(i) a hCMVie virus enhancer consisting of nucleotide sequence SEQ ID NO: 1 and possessing an activity of transcription activation,
(ii) a promoter region of B-actin consisting of nucleotide sequence SEQ ID NO: 3 and possessing a promoter activity, and
(iii) a 5′ UTR region consisting of sequence SEQ ID NO: 10 and having properties of stabilization of mRNA and a translation facilitator.
2. The transcription unit according to claim 1 , wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25, wherein said transcription unit, when expressed in a cell or cell line, is capable of producing a volume-based production of a protein of interest that is greater than that obtained with a transcription unit consisting of a combination of said CMV enhancer together with said promoter region of β-actin.
3. An expression vector comprising at least one transcription unit as defined according to claim 1 and at least one cloning site allowing integration of a nucleic acid coding for a protein of interest.
4. An expression vector comprising at least one transcription unit as defined according to claim 1 and at least one site for site-specific recombination allowing integration of a nucleic acid coding for a protein of interest.
5. The expression vector according to claim 3 , further comprising a eukaryotic resistance gene, a bacterial resistance gene, a bacterial replication origin and a unit dedicated to gene amplification.
6. The expression vector according to claim 3 , wherein said protein of interest is selected from the group consisting of the proteins participating in coagulation, immunoglobulins, cytokines, hormones, growth factors or complement factors and any fusion protein.
7. A host cell comprising an expression vector as defined in claim 3 .
8. The host cell according to claim 7 , wherein said host cell is a CHO-S, a CHO, or an HEK cell line.