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Gene fragment, recombinant vector and use thereof for improving expression level of exogenous gene in mammalian cells

An exogenous gene and gene technology, applied in the field of genetic engineering, can solve the problems of reducing the half-life of mRNA, unfavorable RNA secondary structure, and lack of verification of expression, so as to overcome the position effect, have good application prospects, and reduce losses.

Active Publication Date: 2018-09-18
四川丰讯科技发展有限公司
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  • Abstract
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Problems solved by technology

However, in this literature, the expression vector PDEF38 did not test that the exogenous gene would also amplify with the increase of MTX concentration. We constructed the vector containing the upstream and downstream regulatory sequences of EF1alpha according to the literature. The expression of the source gene has not increased, and it is likely that its 4Kb regulatory sequence contains a sequence similar to an insulator, and the gene that maintains its expression box is not affected by the surrounding environment, resulting in an increase in the concentration of MTX. Only the DHFR gene can be amplified and its downstream sequence, while its upstream EF1alpha regulatory sequence and exogenous gene are not amplified
Orlova and Kovnir et al. (BMC Biotechnology 2014, 14:56) reported using the upstream 4.1Kb and 4.2Kb downstream sequences of the EF1alpha gene to connect the target exogenous gene with the DHFR gene through the IRES between the regulatory sequences to form a dicistronic Expression vector, when using EGFP as the target gene, the expression level of EGFP increased by 8 times and 4.5 times respectively through a round of amplification during adherent culture and suspension culture, indicating that this bicistronic expression vector can Obtaining highly active promoter characteristics can be amplified, but the literature does not verify whether the expression of other target genes can obtain such good results
Some literature (Mcgrew JT. Vectors and methods for recombinant protein expression, Uspatent No: 6632637B1.) reported that two genes connected by IRES form a large mRNA, which may reduce the half-life of mRNA; it is also possible to form an RNA that is not conducive to protein translation. level structure; resulting in decreased expression of the gene of interest

Method used

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  • Gene fragment, recombinant vector and use thereof for improving expression level of exogenous gene in mammalian cells
  • Gene fragment, recombinant vector and use thereof for improving expression level of exogenous gene in mammalian cells
  • Gene fragment, recombinant vector and use thereof for improving expression level of exogenous gene in mammalian cells

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1 Construction of the recombinant vector of the present invention and its effect verification

[0027] 1. Construction method

[0028] (1) Cloning of the transcriptional regulatory sequence of CHO cell EF-alpha1 and the MAR sequence of chicken lysozyme.

[0029] 1. Extraction of CHO cell genomic DNA sequence

[0030] CHO cells were cultured with D / F medium 10% serum until the cell square flask was overgrown, and digested with trypsin. Genomic DNA of CHO cells was extracted according to the operating instructions of Transgen's EasyPure Genomic DNA kit.

[0031] 2. Cloning of the gene transcription regulatory sequence of CHO peptide elongation factor 1

[0032] According to the sequence design of Genbank's report following primers:

[0033] CHEF1:5-ata acgcgt GCAGATCCGT CGAGCTCTCG GCCACCGAGC-3

[0034] CHEF2:5-ata gctagc ACACCTTAAA AAAAAAGTTC GAAGAATACC-3

[0035] CHEF3:5-ata tctaga AATATTACCC CTAACACCTG CCACCCCAGT C-3

[0036] CHEF4:5-ata tccgga AGCAAAGCC...

Embodiment 2

[0061] Example 2 Expression of exogenous protein using the combination carrier CHMARCHEF1506 / DHFR of the present invention

[0062] Example 1 shows that the carrier CHMARCHEF1506DHFR expresses good fluorescence intensity of EGFP, indicating that the gene 5' and 3' end regulatory sequences of CHO endogenous peptide elongation factor 1 and the chicken lysozyme MAR sequence are used to express foreign genes in CHO cells The effect is better, and the exogenous gene aldalimumab is further used to verify the effect of this vector.

[0063] On CHMARCHEF53 / DHFR through the NheI and XhoI restriction sites, the aldalimumab heavy chain gene (the sequence of the aldalimumab heavy chain gene is shown in SEQ ID NO.14) was constructed into the vector CHMARCHEFaldalimumabHC / DHFR ( Figure 5); the aldalimumab light chain gene (the sequence of the aldalimumab light chain gene is shown in SEQ ID NO.15) is also connected into the vector CHMARCHEF1506 / EGFP through the same digestion site through t...

Embodiment 3

[0067] Example 3 Study on the stability of exogenous protein expressed by the combined vector CHMARCHEF1506 / DHFR of the present invention

[0068] The five strains of cells that were highly expressed in the shake flask of Example 2 were obtained by 3x10 5 / ml cell density inoculation, 3-4 days the cells grow to 2 million / ml, and then press 3x10 5 / ml density and have been subcultured in this way for 50-60 days in shake flasks. The results showed that the expression of aldalimumab in the obtained high-expression cell lines did not change significantly after subculture for about 60 days, and the final subculture cells were cultured in serum-free medium supplemented with glucose for 14 days, and the five cell lines expressed The amounts are about 1-1.3 g / L, indicating that the high-expression vector constructed in this way can achieve stable and high-yield expression of foreign proteins in CHO cells.

[0069] According to literature reports, if the culture conditions are optimi...

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Abstract

The invention discloses a gene segment for increasing an exogenous gene expression level in mammalian cells. The gene segment is characterized in that the nucleotide thereof is shown as SEQ ID NO.1 or 2. The invention also discloses a recombinant plasmid. The recombinant plasmid is characterized in that a promoter CMV in pcDNA 3.1(+) plasmid is replaced by the sequence in the length of a 5' terminal of a CHEF-alpha gene of 1543bp, the BGHPA is replaced by the sequence in the length of a 3' terminal of a CHEF-alpha gene of 610bp, the chicken lysozyme MAR sequence is inserted into the upstream side of the promoter, and a selection marker gene Neo is replaced by a DHFR gene. The invention also discloses an application of the recombinant plasmid. The recombinant plasmid disclosed by the invention carries the exogenous gene transfection mammalian cells, the expression level of the exogenous gene is high, the expression level of the exogenous gene can continuously stabilize a high yield, and the application prospect is excellent.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a gene fragment, a recombinant vector and an application thereof for improving the expression level of exogenous genes in mammalian cells. Background technique [0002] Chinese hamster ovary cells (CHO) are the most commonly used mammalian cells in biopharmaceuticals. They are one of the best expression systems for exogenous eukaryotic genes. They are widely used in biopharmaceuticals. They are currently on the market and undergoing clinical research. About 60-70% of engineering drugs are products expressed by mammalian cells, and the products expressed by CHO cells account for the vast majority of them. Compared with the prokaryotic expression system, it has the following advantages: foreign proteins are easily synthesized in CHO cells and secreted into the medium; the folding and modification, physical and chemical properties, and biological properties of recombin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85
CPCC07K14/47C12N15/85C12N2800/107C12N2830/34
Inventor 罗世超罗川
Owner 四川丰讯科技发展有限公司
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