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Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof

A technology of exogenous gene and high-efficiency expression, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve problems such as gene silencing effect, achieve overcoming position effect, increase transcription Intensive, expressive effects

Inactive Publication Date: 2011-11-23
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the CpG island present in the promoter can easily lead to the silencing effect of the gene, and the CMV promoter only functions in the S phase of the cell cycle

Method used

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  • Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof
  • Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof
  • Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Cloning of the MAR sequence of embodiment 1 people β-globin

[0048] Genomic DNA was extracted from HEK293 cells. According to the human β-globin MAR sequence reported by GenBank, which contains a segment of Alu sequence that should be a repetitive sequence in the human genome, the segmented cloning method was used to clone 1-2804bp by PCR. The synthetic method synthesizes 2804-2998bp, and then splicing the two to obtain a human β-globin MAR sequence with a length of 2998bp. Synthesize HUMAR1 and HUMAR2 primers to amplify 2804bp fragments. The amplification conditions are: denaturation at 94°C for 4min, 1min at 94°C, 1min at 68°C, 40sec at 72°C for 2min, 40sec at 72°C, extension at 72°C for 10min, and cooling at 4°C. The DNA polymerase was LA Taq DNA polymerase.

[0049] The sequence of primer HUMAR1 is shown in SEQ ID No.7.

[0050] The sequence of the primer HUMAR2 is shown in SEQ ID No.8.

Embodiment 2

[0051] Example 2 Cloning of hEF-1α Gene Transcription Regulatory Elements

[0052] The following primers were designed according to the sequence reported by Genbank:

[0053] The sequence of the primer HuEF1 is shown in SEQ ID No.9.

[0054] The sequence of the primer HuEF2 is shown in SEQ ID No.10.

[0055] The sequence of the primer HuEF3 is shown in SEQ ID No.11.

[0056] The sequence of the primer HuEF4 is shown in SEQ ID No.12.

[0057] Use primers HuEF1 and HuEF2 to amplify the 4093bp nucleotide sequence of hEF-1α5' including the promoter and intron 1, and the amplified product contains NheI and MluI restriction sites; primers HuEF3 and HuEF4 amplify 4018bp hEF-1α3 The nucleotide sequence at the 'end, the amplified product contains XbaI and BspEI restriction sites. LA Taq DNA polymerase from Treasure Biotech was used. Amplification conditions were 94°C for 5 min, 94°C for 30 sec, 60°C for 30 sec, 72°C for 4 min, 30 cycles, and 72°C for 10 min. The amplified product ...

Embodiment 3

[0058] Cloning of Example 3 Artificial Transcription Factor Binding Site Nucleotide Sequence

[0059] For the convenience of cloning, PacI, AgeI, HpaI restriction site, use these two primers to amplify the artificial transcription factor binding site sequence containing 5 GaL4 binding sites from the plasmid PG5luc (Promega product). Use TAKARA high-fidelity Pybest enzyme to amplify, and recover a fragment of about 150bp. Ligated into pcDNA3.1(+) / MCS vector through PacI and AgeI, connected into pcDNA3.1(+) / UAS through AgeI and HpaI, and named pcDNA3.1(+) / 2UAS after correct sequencing. Cloning of Example 4 Post-transcriptional Regulatory Element WPRE Sequence

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Abstract

The invention discloses a combined vector for mediating the high-efficiency expression of an exogenous gene in cells of a mammal, which comprises an exogenous gene high-efficiency expression vector pATEW and an artificial transcription factor pcDNA3.1(+) / Hy / GVP4. The exogenous gene high-efficiency expression vector pATEW combines gene expression regulatory elements including a human beta-globin MAR sequence, a hEF-1a genetic transcription regulatory sequence, an after-transcription regulatory element WPRE and the like and an artificial transcription factor combined site sequence. The artificial transcription factor expression vector (pcDNA3.1(+) / Hy / GVP4 is an artificial transcription factor GVP4 which is formed by connecting two parts: four tandem repeats of 12 peptides (DALDDFDLDMLG) in VP16, which serves as the functional structural region of the artificial transcription factor; and the nuclear localization sequence of SV40. By co-transfecting cells of the mammal with the pATEW carrying the exogenous gene and the pcDNA3.1(+) / Hy / GVP4, the high-efficiency expression of the exogenous gene in the cells of the mammal can be realized.

Description

technical field [0001] The invention relates to an expression vector, in particular to a vector for mediating high-efficiency expression of foreign genes in mammalian cells and its application. Background technique [0002] Since the tissue-type plasminogen activator (tissue-type plasminogen activator, tPA) was listed as the first recombinant protein drug produced by mammalian cell expression system in 1987, mammalian cell expression system has been used in the production of recombinant pharmaceutical protein. gradually became dominant. At present, 65% to 75% of recombinant protein drugs marketed abroad are produced using mammalian cell expression systems. [0003] A major advantage of mammalian cell expression systems is their ability to perform complex post-translational modifications on protein molecules. These modifications include N-linked and O-linked glycosylation, folding of nascent polypeptides, hydrolysis, and disulfide bond formation, among others. Compared wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/67C12N15/63C12N5/10
Inventor 李世崇陈昭烈叶玲玲刘红刘兴茂何文俊王启伟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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