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New application of protein Arginine methyltransferase 5

An arginine methyl and protein technology, applied to cells modified by introducing foreign genetic material, using a vector to introduce foreign genetic material, fermentation, etc., can solve the problem of less CHO cell lines, cell apoptosis, and foreign protein expression levels. limited issues

Inactive Publication Date: 2011-09-28
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in bioengineering pharmaceuticals, the use of CHO cells to express foreign proteins still has the following technical problems: there are few CHO cell lines stably expressing the target protein; the expression level of foreign proteins is limited; Modification levels such as basement need to be improved, etc.

Method used

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  • New application of protein Arginine methyltransferase 5
  • New application of protein Arginine methyltransferase 5
  • New application of protein Arginine methyltransferase 5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1, Construction of a CHO-K1 cell line stably expressing PRMT5

[0024] 1. Construction of recombinant plasmid pCMV-Flag-PRMT5

[0025] 1. Using the plasmid (Incyte Full Length Human cDNA Clone; Open Biosystems plasmid product, catalog number IHS1380-97430767) containing the full-length sequence of PRMT5cDNA as a template, PCR amplification was carried out to obtain the PCR amplification product (containing the full-length fragment of the PRMT5cDNA sequence , see sequence 2) of the sequence listing.

[0026] The primer pairs for PCR amplification are as follows:

[0027] Upstream primer: 5'-G GAATTC GGCACGAGGGCGAGGAGAAAGATGGCGGCGATGGCG-3' (EcoRI);

[0028] Downstream primer: 5'-CCG CTCGAG CTAGAGGCCAATGGTATATGAGCG-3' (XhoI).

[0029] The annealing temperature for PCR amplification was 56°C.

[0030] 2. Digest the PCR amplification product with restriction endonucleases EcoRI (TAKARA, catalog number D1010A) and XhoI (TAKARA, catalog number D1094A), and reco...

Embodiment 2

[0045] Embodiment 2, the proliferation situation of CHO-PRMT5

[0046] The same number of cells (5~8×10 4 ), CHO-K1 cells (CHO), CHO-2B cells (2B) and three different CHO-PRMT5 cell lines (7A5, 8C2 and 1C4) were evenly spread in Φ60mm plates, and medium A (containing 5 % volume percent calf serum) and medium B (containing 0.5% volume percent calf serum) for culture. Three replicate dishes were set up for each culture treatment, and the results were statistically averaged. 24hr, 48hr and 72hr after plate plating were taken and the number of cells was counted. Cell growth was quantitatively analyzed hourly by the MTT assay.

[0047] The results of MTT analysis when medium A is used are shown in figure 2 . For photos of cells using medium B, see image 3 . In culture medium A (5% calf serum), there was no significant difference in cell expansion observed under a microscope, and the quantitative analysis results of MTT method also showed that the cell growth rate was almos...

Embodiment 3

[0048] Example 3, application of CHO-PRMT5 cell line to express IL2 protein

[0049] 1. Preparation of recombinant plasmid IL2-pEGFP-N3

[0050] 1. PCR amplification of the insert

[0051] Using the plasmid containing the full-length sequence of IL2 cDNA (Incyte Full Length Human cDNA Clone; Open Biosystems plasmid product, catalog number IHS1380-97431972) as a template, perform PCR amplification to obtain PCR amplification products (containing The full-length fragment of the IL2 cDNA sequence; the DNA shown in the 1st to 459th nucleotides from the 5' end of the sequence 4 of the sequence listing).

[0052] The primer pairs for PCR amplification are as follows:

[0053] Upstream primer: 5'-CCC AAG CTT GCC ACC ATG TAC AGG ATG CAA CTC C-3';

[0054] Downstream primer: 5'-CG GA ATT C CA AGT CAG TGTTGAGATGATGC-3'.

[0055] 2. Digest the PCR amplification product of step 2 with restriction endonucleases HindIII and EcoRI, and recover the target fragment of about 470bp.

...

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Abstract

The invention discloses a new application of protein Arginine methyltransferase 5. The new application provided by the invention is that protein Arginine methyltransferase 5 and the coding gene thereof are utilized to promote mammalian cells to express foreign proteins. The invention protects a method for preparing a recombinant mammalian cell line expressing foreign proteins. The method is as follows: the coding gene of Arginine methyltransferase 5 is introduced in the mammalian cells to obtain the recombinant mammalian cell line expressing foreign proteins. The invention also protects the recombinant mammalian cell line expressing foreign proteins prepared by the method. The recombinant cell line prepared by the method can be utilized to efficiently express the drug protein modified by glycosylation, the cell line has higer efficiency and lower demand on the purification process; and when applied in the production of bio-engineering drugs, the cell line has obvious economic benefit.

Description

technical field [0001] The present invention relates to a new application of protein arginine methyltransferase 5, in particular to the application of protein arginine methyltransferase 5 in improving protein production and secretion efficiency of mammalian cell lines. Background technique [0002] 1. Technological progress of expressing foreign proteins in mammalian cell lines [0003] The prokaryotic system for expressing recombinant proteins through Escherichia coli is still a common system for large-scale expression of foreign proteins. However, in the face of proteins with large molecular weight, complex structure, many disulfide bonds, and activity-determined modification groups, the prokaryotic system has been difficult to achieve. With the in-depth study of eukaryotic gene expression and regulation, it has been proved that yeast can also be a useful expression system. However, the yeast system also has many shortcomings, such as complicated purification process, hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12P21/02
Inventor 鲍时来邹振华周忠卫
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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