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Expression vector for efficiently expressing exogenous gene of HEK293 cell

A high-efficiency expression technology of exogenous genes, applied in the direction of introducing foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc. by using vectors, can solve the problems of silencing effect, weak expression ability not as good as CMV promoter, etc., to promote high-efficiency expression Effect

Inactive Publication Date: 2010-10-20
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, studies have found that the presence of CpG islands in this promoter can easily lead to silencing effects; it only works in the S phase of the cell cycle, so it is necessary to find a better promoter than CMV
People have been looking for a promoter with high strength and wide adaptability. Kalwy S uses the mouse mCMV promoter to express GFP in CHO-K1 cells, which is 3 times that of the human CMV promoter, but when expressing proteins such as antibodies, The expression ability is weaker than human CMV promoter

Method used

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  • Expression vector for efficiently expressing exogenous gene of HEK293 cell
  • Expression vector for efficiently expressing exogenous gene of HEK293 cell
  • Expression vector for efficiently expressing exogenous gene of HEK293 cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cloning of gene transcriptional regulatory sequence of human peptide elongation factor 1.

[0031] 1. Extraction of genomic DNA sequence of HEK293 cells

[0032]HEK293 cells were cultured in D / F medium with 5% serum until the cell square flask was overgrown, and digested with trypsin. Genomic DNA of HEK293 cells was extracted according to the operation manual of TAKARAGeneBall Genome preparation kit.

[0033] 2. Cloning of the gene transcriptional regulatory sequence of human peptide elongation factor 1

[0034] The following primers were designed according to the sequence reported by Genbank, as shown in SEQ ID No.3-6:

[0035] HuEF1 (SEQ ID No. 3): 5-ata acgcgt CCCAAAACAC ATTTACGAGC CTCAACATCGTTACTG-3

[0036] HuEF2 (SEQ ID No. 4): 5-ata gctagc TCA CGACACCTGA AATGGAAGAA AAAAACTTTGAACC-3

[0037] HuEF3 (SEQ ID No. 5): 5-ata tctaga ATATTATCC CTAATACCTG CCACCCCACTCTTAATCAGT GG-3

[0038] HuEF4 (SEQ ID No. 6): 5-ata tccgga CATTAAAAAG TAAGAGATTA CTGATATATACA...

Embodiment 2

[0040] Example 2 Construction of Vectors Containing Different Sequences of Human Peptide Elongation Factor 1

[0041] 1. Reconstruct pCDNA3.1(+)

[0042] Synthesize the following primers:

[0043] BGH1 (SEQ ID No. 7): 5-TCGAGTCTAGAGGGCCCGTTTAAAC-3

[0044] BGH2 (SEQ ID No. 8): 5-ATATCCGGACTCAGAAGCCATAGAGCCCACC-3

[0045] FOR1 (SEQ ID No.9): 5-ATATCCGGAGCGGAAAGAA CCAGCTGGGG-3

[0046] FOR2 (SEQ ID No. 10): 5-TATACAAGCTCCCGGGAGCTTTTTGC-5

[0047] Primer BGH1 contains an XbaI restriction site, BGH2 contains a BspEI restriction site, FOR1 contains a BspEI restriction site, and FOR2 contains an XmaI restriction site. Using pCDNA3.1(+) as a template by PCR method, 200bp and 800bp fragments were respectively amplified. After gel recovery, the 200bp fragments were digested with XbaI and BspEI respectively, the 800bp fragments were digested with BspEI and XmaI, and the 800bp fragments were digested with XbaI and XmaI. After enzyme digestion, the large fragments were recovered from...

Embodiment 3

[0050] Example 3 Vector Transfection of HEK293 Cells and the Measurement of the Fluorescent Expression Intensity of the Reporter Gene EGFP

[0051] One day before transfection, HEK293 cells were treated with 7.5×10 5 Cells were plated in a 6-well plate, and the cells were cultured overnight, and the expression vectors pcDNA3.1 / EGFP and pcDNAPEF / EGFP, pcDNA HEF5UTR / EGFP, pcDNAHEF5UTR / EGFP / HEF3UTR, pcDNACMV / EGFP / HEF3UTR were transfected into HEK293 cells at an equimolar ratio , transfection reagent with Lipofectamine TM In 2000, the operation method was carried out according to the instructions of the reagents.

[0052] 24 hours after transfection, the cells were digested with trypsin, and a 6-well plate was inoculated into a 24-well plate. After 24 hours, after the cells adhered to the wall, the medium was replaced with antibiotic G418 0.3mg / ml. Change the fluid every 3-4 days. Wait for positive clones to grow out after 2 weeks. Trypsinize, collect positive cells, centrifug...

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Abstract

The invention discloses a method for constructing a vector for efficiently expressing an exogenous gene in an HEK293 cell, which is used for constructing a nucleotide sequence of a transcription regulatory sequence of the carrier. The expressions of expressing exogenous gene EGFP, proUK and tPA by using the constructed efficient expression vector are 7, 5 and 2 times those of using a comparison vector respectively; and the expression vector combining with artificial transcription factors can promote the expression of the EGFP, and the expression can be improved by 15 times compared with PcDNA / 3.1 / EGFP.

Description

technical field [0001] The invention relates to an expression vector for highly expressing exogenous genes in HEK293 cells. Specifically, the transcription control sequence of the housekeeping gene peptide elongation factor of HEK293 cells is used to promote the high-efficiency expression of foreign genes in HEK293 cells. Background technique [0002] HEK293 cells are immortalized human embryonic kidney epithelial cell lines constructed by Graham et al. in 1977. Because of its good post-translational modification ability, it is widely used in the functional research of many human proteins, and it is also widely used as a packaging host for viruses. In the field of cell engineering, HEK293 cells have also been used, for example to produce human protein C. Some enzymes unique to human host cells help to fold recombinant proteins into the correct conformation to improve their biological activity. For example, there is α-2,6 sialyltransferase in human host cells, which can mo...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/66C12N15/11
Inventor 李世崇陈昭烈叶玲玲刘红吴本传刘兴茂王启伟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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