CN102199623A - New application of protein Arginine methyltransferase 5 - Google Patents
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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
The present invention relates to the new purposes of Protein Arginine Methyltransferase 5, particularly relate to Protein Arginine Methyltransferase 5 in protein output that improves mammal cell line and the application aspect the secernment efficiency.
Background technology
One, mammal cell line is expressed the technical progress of foreign protein
Prokaryotic system by express recombinant proteins such as intestinal bacteria remains the customary systems that foreign protein is expressed in mass-producing so far.But, complex structure big in the face of molecular weight, disulfide linkage are many, modification group determines active protein, and the protokaryon system has been difficult to realization.Along with going deep into of eukaryotic gene expression and study on regulation, prove that yeast also can become useful expression system, yet also there are a lot of shortcomings in the yeast system, do not reach requirement, poor activity etc. as purifying technique complexity, cost height, goods purity.
Along with increase to the market requirement of proteinaceous product that characteristics such as modifying complexity, high reactivity are arranged; become industrial trends of the times such as bio-pharmaceuticals by mammalian cell mass-producing expression and production protein; accounted for more than 80% of biological-pharmacy, the annual output value is above 20,000,000,000 dollars.With respect to procaryotic cell expression, mammalian cell expression is more suitable for producing that molecular weight is big, complex structure, disulfide linkage are many, modification group determines active protein, as monoclonal antibody and enzyme etc.With respect to the yeast system, the protein of mammalian cell expression is at chance processed after the translation more (as occur on the golgi body glycosylation), can promote product correctly folding, form highly active configuration, the vaccine product by its processing has the immunogenicity with respect to 16～20 times on yeast type.In addition, mammalian cell is also easily by the recombinant dna plasmid transfection, and has genetic stability and repeatability, and the mammalian cell through transforming can be secreted into expressed products in the substratum, and purifying technique is simple, cost is low.In view of above-mentioned advantage, the mammalian cell strain of countries in the world employing long-term cultivation was just advised by biological products research group of The World Health Organization (WHO) in 1986, produce various biological products.At present, American-European countries is the widespread use mammalian cell, as Chinese mouse oophoroma cell (CHO-K1, Chinese Hamster Ovary), produces products such as pharmaceutical grade protein, recombinant vaccine, activated protein and polypeptide.
The CHO-K1 cell is to use maximum mammalian cell expression systems in the present biological-pharmacy.It is an isolating strain epithelial cell from Chinese hamster ovary, and it lacks Tetrahydrofolate dehydrogenase, and (dehydrofolate reductase negative, mutant strain dhfr-) are one of most widely used mammalian gene expression recipient cells.First biologics t-PA that gets permission to use adopts CHO (dhfr-) system to produce.At present, existing various exogenous genes is as the expression of succeeding in CHO (dhfr-) cell of tens kinds of bio-pharmaceuticals such as erythropoietin (EPO), interferon-, IFN-, the curative antibody of multiple reorganization and genetic vaccine.This cell has following advantage: foreign gene is not having to stablize maintenance under the situation of selective pressure; Be fit to the secreting, expressing and the interior expression of born of the same parents of multiple proteins; Requirement to substratum is lower, can cultivate in serum free medium; Cell can carry out adherent culture and extensive suspension culture.Yet in biotech medicine product, utilize the expressing cho cell foreign protein still to have following technical problem: the Chinese hamster ovary celI strain of stably express target protein is less; The exogenous protein expression level is limited; The cell generation apoptosis that goes down to posterity for a long time; Modification levels such as protein glycosylation have much room for improvement etc.This also reflects the two big key issues of utilizing mammalian cell to produce biologics: the one, cross over the technical matters that animal cell large-scale is cultivated; The 2nd, improve obtaining producing and secretory protein through engineering approaches mammal cell line more efficiently.
The height of the expression amount of external source pharmaceutical protein in mammalian cells such as CHO, except the influence that is subjected to genetic transcription and translation skill, the regulation and control after also being translated.For a long time the improvement of engineering cell system is substantially only paid close attention to the former, and in fact the latter is also very important.The low reason of most of foreign protein expression amount in eukaryotic cell mainly is because expressed albumen can not carry out correct modification and folding on golgi body, and these great expression but the incorrect foreign protein of structure are degraded in tenuigenin.
Two, Protein Arginine Methyltransferase 5 (PRMT5)
The protein arginine methylated transferase, promptly PRMT family has found 10 members at mammalian cell at present, is respectively PRMT1-10.Specificity according to substrate and reaction product can be divided into two classes with these 10 enzymes.Two zymoid common features are can both catalytic proteins arginine generation monomethylation.Two zymoid differences are: two the methylating of class energy catalytic proteins arginine generation asymmetry; And two the methylating of another kind of energy catalysis arginine generation symmetry.PRMT5 and PRMT7 belong to second class, catalysis arginine guanine group generation monomethylation and symmetric two methylating, and other 8 enzymes all belong to the first kind, catalysis arginine guanine group generation monomethylation and asymmetric two methylating.PRMT5 claims JBP1, Skb1Hs or Hsl7 again, separate to obtain by yeast two-hybrid the earliest, in eukaryote from the yeast to the Mammals its protein in evolution camber homology and conservative.
Summary of the invention
The purpose of this invention is to provide a kind of Protein Arginine Methyltransferase 5 (Protein Arginine Methyltransferase 5, new purposes PRMT5).
New purposes provided by the invention is the application in promoting the mammalian cell expression foreign protein that exsomatizes of Protein Arginine Methyltransferase 5 or its encoding gene.Described mammalian cell can be the mammalian cell outside the human embryo stem cell.Described mammalian cell specifically can be mammal cell line.Described mammalian cell more specifically can be Chinese hamster ovary cell (CHO-K1 clone).Described Protein Arginine Methyltransferase 5 can be the protein (GenBank Accession Number:NP_006100 is from N-terminal the 1st to 637 amino acids residue) shown in the sequence 1 of sequence table.The encoding gene of described Protein Arginine Methyltransferase 5 can be the dna molecular shown in the sequence 2 of sequence table (GenBank Accession Number:NM_006109 (GeneID:10419) is from 5 ' terminal the 1st to 1914 Nucleotide).Described foreign protein specifically can be the IL2 albumen (GenBank Accession Number:NP_000577 (GeneID:3558) is from N-terminal the 1st to 153 amino acids residue) shown in the sequence 3 of sequence table.
The present invention also protects a kind of method for preparing reconstitution cell, is the mammalian cell that the encoding gene importing of Protein Arginine Methyltransferase 5 is exsomatized, and obtains reconstitution cell; The function of described reconstitution cell is as the host cell of expressing foreign protein.Described mammalian cell can be the mammalian cell outside the human embryo stem cell.Described mammalian cell specifically can be mammal cell line.Described mammalian cell more specifically can be Chinese hamster ovary cell (CH0-K1 clone).Described Protein Arginine Methyltransferase 5 can be the protein shown in the sequence 1 of sequence table.The encoding gene of described Protein Arginine Methyltransferase 5 can be the dna molecular shown in the sequence 2 of sequence table.The encoding gene of described Protein Arginine Methyltransferase 5 specifically can import described mammalian cell by recombinant plasmid pCMV-Flag-PRMT5; Described recombinant plasmid pCMV-Flag-PRMT5 specifically can be the recombinant plasmid that obtains at the dna molecular shown in the sequence 2 of the multiple clone site insertion sequence table of pCMV-Tag2B plasmid.Described foreign protein specifically can be the IL2 albumen shown in the sequence 3 of sequence table.
The reconstitution cell for preparing with aforesaid method also belongs to protection scope of the present invention.
The application of described reconstitution cell in expressing foreign protein also belongs to protection scope of the present invention.Described foreign protein specifically can be the IL2 albumen shown in the sequence 3 of sequence table.
The present invention also protects a kind of method of expressing foreign protein, is to carry out cell cultures after encoding gene with described foreign protein imports described reconstitution cell, obtains foreign protein.Described foreign protein specifically can be the IL2 albumen shown in the sequence 3 of sequence table.The encoding gene of described foreign protein specifically can be the sequence 4 of sequence table from the dna molecular shown in 5 ' terminal the 1st to 459 Nucleotide (GenBank Accession Number:NM_000586 is from 5 ' terminal the 1st to 459 Nucleotide).Described foreign protein specifically can import described reconstitution cell by recombinant plasmid I L2-pEGFP-N3; Described recombinant plasmid I L2-pEGFP-N3 specifically can be the recombinant plasmid that obtains from the dna molecular shown in 5 ' terminal the 1st to 459 Nucleotide at the multiple clone site insertion sequence 4 of pEGFP-N3 carrier.
In mammalian cell, organoids such as endoplasmic reticulum and golgi body are the important places that protein folds and modifies.This class organoid is implemented to transform; be expected to solve because insufficient modification of translation product and incorrect folding and, make every effort to the protein " work in-process " that mammalian cell translates in a large number and in follow-up processing, transport process, reach high-level efficiency equally the bottleneck effect that the large-scale production foreign protein causes.The present invention makes up the CHO-K1 cell strain that has obtained stably express PRMT5.In the normal serum substratum, the ability that the CHO-PRMT5 cell strain is expressed IL2-GFP is significantly higher than strain of empty carrier control cells and Chinese hamster ovary celI strain.The energy for growth of CHO-PRMT5 cell strain under low serum condition is better than strain of empty carrier control cells and Chinese hamster ovary celI strain.The CHO-K1 cell strain can efficiently express IL2 etc. and have glycosylation modified pharmaceutical protein, and efficient is higher, and is lower to the requirement of purifying process, is applied to the biotechnology drug manufacture and can produces obvious economic benefit.
Description of drawings
Fig. 1 is the partial results that Western detects among the embodiment 1.Stably express Flag-PRMT5 fusion rotein as positive control behind the P:CHO-K1 cell transfecting recombinant plasmid; The N:CHO-K1 cell; 5A1,6B4,8A3,8C2,8B5,5A2,7A5,8D4,8D2,8B5,5B4,5C1,6C3,6C5,4B5,5B2,7C2 are the monoclonal cell strain that obtains behind the CHO-K1 cell transfecting recombinant plasmid.
Fig. 2 is MTT analytical results when adopting culture medium A among the embodiment 2 (D of the unit representative of X-coordinate hour).
Fig. 3 is the photo of cell when adopting substratum B among the embodiment 2.
Fig. 4 uses the 7A5 cell strain to express the proteic Western detected result of IL2 among the embodiment 3.
Fig. 5 uses a plurality of cell strains to express the proteic Western detected result of IL2 among the embodiment 3.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Embodiment all adopts the common DNA product purification test kit (catalog number (Cat.No.) DP204-02) of day root biochemical technology (Beijing) company and plain agar sugar gel DNA to reclaim recovery and the purifying that test kit (catalog number (Cat.No.) DP209-02) carries out DNA.The basic medium of cell cultures is the DMEM substratum in following examples.Flag monoclonal antibody: Sigma, F3165.GFP monoclonal antibody: SANTA CRUZ, sc-9996.Tubulin monoclonal antibody: Sigma, T5168.Actin monoclonal antibody: Sigma, A5060.PEGFP-N3 carrier: BD Biosciences Clontech, catalog number (Cat.No.) 6080-1.PCMV-Tag2B plasmid: Aligent technologies/Genomics company plasmid product, catalog number (Cat.No.) 211172.Intestinal bacteria Top10; The Invitrogen of company, article No. C4040-10.PBMC cell: PBMC kit, the 3H Biomedical AB of company; Article No. 3H14-10.CHO-K1 clone: cell resource center of Shanghai Sheng Ke institute of the Chinese Academy of Sciences, catalog number (Cat.No.) GNHa 7.
The prescription of PBS (0.1M phosphate buffered saline buffer): with 800ml dissolved in distilled water 8g NaCl, 0.2g KCl, 1.44g Na
2HPO
4, 0.24g KH
2PO
4With the pH value to 7.4 of HCl regulator solution, adding distil water is settled to 1L.After the packing at 15psi (1.05kg/cm
2) high pressure steam sterilization 20 minutes, or filtration sterilization, be stored in room temperature.
The CHO-K1 cell strain of embodiment 1, structure stably express PRMT5
One, the structure of recombinant plasmid pCMV-Flag-PRMT5
1, plasmid (the Incyte Full Length Human cDNA Clone to contain the PRMT5cDNA full length sequence; Open Biosystems company plasmid product, catalog number (Cat.No.) IHS1380-97430767) be template, carry out pcr amplification, obtain pcr amplification product (contain PRMT5cDNA sequence full length fragment, see the sequence 2 of sequence table).
The primer of pcr amplification is to as follows:
Upstream primer: 5 '-G
GAATTCGGCACGAGGGCGAGGAGAAAGATGGCGGCGATGGCG-3 ' (EcoRI);
Downstream primer: 5 '-CCG
CTCGAGCTAGAGGCCAATGGTATATGAGCG-3 ' (XhoI).
The annealing temperature of pcr amplification is 56 ℃.
2,, reclaim the purpose band of about 1.9kb behind the electrophoresis with restriction enzyme EcoRI (TAKARA, catalog number (Cat.No.) D1010A) and XhoI (TAKARA, catalog number (Cat.No.) D1094A) double digestion pcr amplification product.
3, with restriction enzyme EcoRI and XhoI double digestion pCMV-Tag2B plasmid, electrophoresis reclaims carrier framework (about 4.9kb).
4, the purpose band that step 2 is reclaimed is connected with TAKARA T4 DNA Ligase with the carrier framework that step 3 reclaims, and connection product transformed into escherichia coli Top10 competent cell (will connect product and competent cell at mixing on ice, ice bath 30 minutes; 42 ℃ of heat shock 90s are placed on ice rapidly, ice bath 5min; Add the liquid LB substratum of 800 μ l, 37 ℃ of 100rpm are hatched 45min), be coated onto on the LB flat board (containing the 50ug/ml kantlex) 37 ℃ of overnight incubation.
5, picking positive colony, 37 ℃ are shaken bacterium and spend the night in liquid LB substratum, collect thalline, the extraction plasmid carries out enzyme with restriction enzyme EcoRI and XhoI and cuts evaluation, and the clone that enzyme is cut product electrophoresis showed 4.9kb and 1.9kb two bands cuts the positive colony of evaluation for enzyme.
6, enzyme is cut evaluation male cloning and sequencing, pCMV-Flag-PRMT5 successfully constructs by sequencing result conclusive evidence recombinant plasmid.The structrual description of recombinant plasmid pCMV-Flag-PRMT5: the sequence 2 of having inserted sequence table between the EcoRI of pCMV-Tag2B plasmid and XhoI restriction enzyme site is from the DNA shown in 5 ' terminal the 1st to 1914 Nucleotide; The male recombinant plasmid can be used for expressing the Flag-PRMT5 fusion rotein.
Two, make up the CHO-K1 cell strain of stably express PRMT5
1, the transfection of plasmid
The CHO-K1 cell evenly is layered in the Φ 35mm plate, treat that 24 hour cells are adherent after, use Lipofectamine2000 transfection 1 μ g recombinant plasmid pCMV-Flag-PRMT5.
2, G418 screening stable expression cell line
Behind the transfection plasmid 24 hours, use trypsin digestion cell, be laid on (three gradients: 1000,500,100 in every ware cell) in the Φ 60mm plate with rarer density again.The G418 (1200ug/ml) that adds primary dcreening operation concentration in the substratum.Changed the fresh culture that contains G418 every three days.Initial several days visible dead and buoyant cells of sieve under the G418 drug effect in a large number after treating not sieve the death situation condition substantially, are adjusted to G418 and are kept with concentration (400ug/ml).
After 14 days, the positive cell of anti-G418 has formed the clone of big or small suitable picking.Remove substratum,, be immersed among the PBS again with the not adherent cell of the careful flush away of PBS.In gnotobasis, draw monoclonal cell group by microscope, be transferred in 24 orifice plates that contain the 50ul pancreatin.Trysinization dissociated cell group behind 3～5min, adds the 500ul substratum, and cell mass is opened in piping and druming gently, allows cell evenly be layered in the hole.Be transferred to 37 ℃ then, 5%CO
2Incubator in continue to cultivate.The clone that about 100 positive cells of picking form carries out enlarged culturing.
When treating that cell covers with in 24 orifice plates, trysinization and enlarged culturing are to Φ 35mm, Φ 60mm plate.Former culture hole (or ware) still stays a little cell to continue to cultivate backup when enlarging.Carry out Western behind the cell harvesting albumen in the Φ 35mm ware and detect if correctly expressed the positive cell of Flag-PRMT5, the cell in its corresponding Φ 60mm ware carries out frozen.Positive cell clone carry out going down to posterity for 4～10 times cultivate after, detect Flag-PRMT5 Expression of Fusion Protein level by Western once more.During Western detected, applied sample amount was 50 μ g total proteins, and one anti-ly is the Flag monoclonal antibody, and two anti-ly are the horseradish peroxidase sheep anti-mouse igg antibody.
Screened the Chinese hamster ovary celI of about 100,000 transfection PRMT5 genes altogether, identified about mono-clonal more than 100, obtained the Chinese hamster ovary celI strain (CHO-PRMT5) of 17 stable expression of exogenous PRMT5 (Flag-PRMT5) by Western Blot.
With pCMV-Tag2B plasmid transfection CHO-K1 cell, method is the same, obtains negative control cell strain (CHO-2B).
The partial results that Western detects is (the albumen applied sample amount is 50 micrograms) as shown in Figure 1.Monoclonal cell strain 5A1,5C1,6B4,8A3,8C2,1C4,8B5,5A2,7A5,8C2,8B5,8D2,8D4 and 5B4 all can stably express Flag-PRMT5.
The propagation situation of embodiment 2, CHO-PRMT5
With same cell quantity (5～8 * 10
4) CHO-K1 cell (CHO), CHO-2B cell (2B) the CHO-PRMT5 cell strain (7A5,8C2 and 1C4) different with three evenly be layered on respectively in the Φ 60mm plate, adopt culture medium A (containing 5% volumn concentration calf serum) and substratum B (containing 0.5% volumn concentration calf serum) to cultivate respectively.Each is cultivated to handle three repetition wares is set, and result's statistics is averaged.24hr behind the ware of shop, 48hr and 72hr statistics are taken pictures and cell quantity.Per hour by mtt assay quantitative analysis cell growth condition.
The MTT analytical results is seen Fig. 2 when adopting culture medium A.The photo of cell is seen Fig. 3 when adopting substratum B.In culture medium A (5% calf serum), the amplification of microscopically observation of cell does not have significant difference, mtt assay quantitative analysis results to show that the cell speed of growth is almost consistent yet; In substratum B (0.5% calf serum), CHO-2B cell and the growth of CHO-K1 cell are suppressed, and the cell amplification speed of cell strain 7A5, the 8C2 of three stably express PRMT5 and 1C4 is not subjected to obvious influence.The result shows: stably express PRMT5 can promote cell at low serum even grow under serum-free condition, and this characteristic mammalian cell expression system just is needed.
Embodiment 3, application CHO-PRMT5 cell strain are expressed IL2 albumen
One, the preparation of recombinant plasmid I L2-pEGFP-N3
1, pcr amplification inserts fragment
With plasmid (the Incyte Full Length Human cDNA Clone that contains IL2 cDNA full length sequence; Open Biosystems company plasmid product, catalog number (Cat.No.) IHS1380-97431972) be template, carry out pcr amplification, obtain the pcr amplification product (fragment that contains the IL2cDNA sequence total length except terminator codon; The sequence 4 of sequence table is from the DNA shown in 5 ' terminal the 1st to 459 Nucleotide).
The primer of pcr amplification is to as follows:
Upstream primer: 5 '-CCC
AAG CTTGCC ACC ATG TAC AGG ATG CAA CTC C-3 ';
Downstream primer: 5 '-CG
GA ATT CCA AGT CAG TGTTGAGATGATGC-3 '.
2, with the pcr amplification product of restriction enzyme HindIII and EcoRI double digestion step 2, reclaim the purpose fragment about 470bp.
3, with restriction enzyme HindIII and EcoRI double digestion pEGFP-N3 carrier, reclaim carrier framework (about 4.7kb).
4, the purpose fragment that step 3 is reclaimed is connected with the carrier framework that step 4 reclaims, the connection product that obtains carries out enzyme successively and cuts evaluation (restriction enzyme HindIII and EcoRI, the purpose fragment is 4.7kb and 470bp) and the order-checking evaluation, the result shows, has obtained recombinant plasmid I L2-pEGFP-N3 (IL2-GFP expression plasmid).The structrual description of recombinant plasmid I L2-pEGFP-N3: the sequence 4 of having inserted sequence table between the HindIII of pEGFP-N3 carrier and EcoRI restriction enzyme site is from the DNA shown in 5 ' terminal the 1st to 459 Nucleotide; This recombinant plasmid can be used for expressing the IL2-GPF fusion rotein.
Two, use the 7A5 cell strain and express IL2 albumen
CHO-K1 cell (CHO), CHO-2B cell (2B) and the same density of CHO-PRMT5 cell strain (7A5) are laid in the Φ 35mm plate, respectively the recombinant plasmid I L2-pEGFP-N3 of transfection equivalent.Cell cultures adopts the substratum that contains 5% (volumn concentration) calf serum.Transfection is harvested cell and substratum after 48 hours, and Western Blot detects IL2-GFP Expression of Fusion Protein amount respectively; Sds polyacrylamide gel electrophoresis: 5% stacking gel, 10% separating gel; One anti-is the GFP monoclonal antibody.Western Blot is confidential reference items with Tubulin, and it is the Tubulin monoclonal antibody that one of detection Tubulin resists.
The results are shown in Figure 4.CHO-PRMT5 cell strain (7A5) is expressed and excretory IL2-GFP raises to some extent than CHO-K1 cell, particularly is secreted into extracellular IL2-GFP.CHO-2B cell expressing and excretory IL2-GFP and CHO-K1 cell basically identical.Express and the equal basically identical of excretory Tubulin in each cell strain.
Three, use a plurality of cell strains and express IL2 albumen
CHO-K1 cell (CHO), CHO-2B cell (2B) CHO-PRMT5 cell strain (7A5,8C2 and 1C4) the same density different with three are laid in the Φ 35mm plate, respectively the recombinant plasmid I L2-pEGFP-N3 of transfection equivalent.Cell cultures adopts the substratum that contains 5% (volumn concentration) calf serum.Transfection is harvested cell and substratum after 48 hours, and ultrasonic (amplitude 40%, ultrasonic time are 5 seconds) lysing cell extracts protein.IL2-GFP fusion rotein and Flag-PRMT5 Expression of Fusion Protein amount in protein that Western Blot detection lysing cell obtains and the substratum; Sds polyacrylamide gel electrophoresis: 5% stacking gel, 10% separating gel; It is the GFP monoclonal antibody that one of detection IL2-GFP fusion rotein resists; It is the Flag monoclonal antibody that one of detection Flag-PRMT5 fusion rotein resists.Western Blot is confidential reference items with Tubulin and Actin; It is the Tubulin monoclonal antibody that one of detection Tubulin resists; It is the Actin monoclonal antibody that one of detection Actin resists.
The results are shown in Figure 5.In three CHO-PRMT5 cell strains, all can detect the Flag-PRMT5 fusion rotein, CHO-K1 cell and CHO-2B cell can not detect the Flag-PRMT5 fusion rotein.In three CHO-PRMT5 cell strains, the expression amount of IL2-GFP and secretory volume all are higher than the CHO-K1 cell, the expression amount of IL2-GFP and secretory volume and CHO-K1 cell basically identical in the CHO-2B cell.In Flag-PRMT5 fusion protein expression high cell strain 8C2 and 1C4, the expression amount of IL2-GFP and secretory volume are higher than cell strain 7A5.Express and excretory Tubulin and the equal basically identical of Actin in each cell strain.The result shows: there is dependency in the PRMT5-CHO cell strain to exogenous protein expression and excretory facilitation effect and PRMT5 expression amount.
Claims (10)
1. Protein Arginine Methyltransferase 5 or its encoding gene application in promoting the mammalian cell expression foreign protein that exsomatizes.
2. application as claimed in claim 1 is characterized in that: described Protein Arginine Methyltransferase 5 is the protein shown in the sequence 1 of sequence table; The encoding gene of described Protein Arginine Methyltransferase 5 is preferably the dna molecular shown in the sequence 2 of sequence table;
Described mammalian cell is the mammalian cell outside the human embryo stem cell; Described mammalian cell is preferably Chinese hamster ovary cell.
3. application as claimed in claim 1 or 2 is characterized in that: described foreign protein is the IL2 albumen shown in the sequence 3 of sequence table.
4. a method for preparing reconstitution cell is the mammalian cell that the encoding gene importing of Protein Arginine Methyltransferase 5 is exsomatized, and obtains reconstitution cell; The function of described reconstitution cell is as the host cell of expressing foreign protein.
5. method as claimed in claim 4 is characterized in that: the encoding gene of described Protein Arginine Methyltransferase 5 is the dna molecular shown in the sequence 2 of sequence table; Described mammalian cell is the mammalian cell outside the human embryo stem cell.
6. method as claimed in claim 5 is characterized in that: the encoding gene of described Protein Arginine Methyltransferase 5 imports described mammalian cell by recombinant plasmid pCMV-Flag-PRMT5; Described recombinant plasmid pCMV-Flag-PRMT5 is the recombinant plasmid that obtains at the dna molecular shown in the sequence 2 of the multiple clone site insertion sequence table of pCMV-Tag2B plasmid; Described mammalian cell is a Chinese hamster ovary cell.
7. the reconstitution cell that arbitrary described method prepares in the claim 4 to 6.
8. a method of expressing foreign protein is to carry out cell cultures behind the described reconstitution cell of encoding gene importing claim 7 with described foreign protein, obtains foreign protein.
9. method as claimed in claim 8 is characterized in that: described foreign protein is the IL2 albumen shown in the sequence 3; The proteic encoding gene of described IL2 is preferably the sequence 4 of sequence table from the dna molecular shown in 5 ' terminal the 1st to 459 Nucleotide.
10. method as claimed in claim 9 is characterized in that: described foreign protein imports described reconstitution cell by recombinant plasmid I L2-pEGFP-N3; The recombinant plasmid that described recombinant plasmid I L2-pEGFP-N3 obtains from the dna molecular shown in 5 ' terminal the 1st to 459 Nucleotide for the sequence 4 at the multiple clone site insertion sequence table of pEGFP-N3 carrier.
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| CN106536511A (en) * | 2014-06-25 | 2017-03-22 | 葛兰素史密斯克莱知识产权发展有限公司 | Crystalline salts of (S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1h)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide |
| CN111235182A (en) * | 2018-11-29 | 2020-06-05 | 中国科学院大连化学物理研究所 | Method for constructing PRMT7 high-expression cell line and cell line |
| CN111542523A (en) * | 2017-11-24 | 2020-08-14 | 朱比连特埃皮斯科瑞有限责任公司 | Heterocyclic compounds as PRMT5 inhibitors |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106536511A (en) * | 2014-06-25 | 2017-03-22 | 葛兰素史密斯克莱知识产权发展有限公司 | Crystalline salts of (S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1h)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide |
| CN106536511B (en) * | 2014-06-25 | 2019-04-30 | 葛兰素史密斯克莱知识产权发展有限公司 | The crystal salt of compound |
| CN111542523A (en) * | 2017-11-24 | 2020-08-14 | 朱比连特埃皮斯科瑞有限责任公司 | Heterocyclic compounds as PRMT5 inhibitors |
| CN111542523B (en) * | 2017-11-24 | 2024-03-29 | 朱比连特埃皮斯科瑞有限责任公司 | Heterocyclic compounds as PRMT5 inhibitors |
| CN111235182A (en) * | 2018-11-29 | 2020-06-05 | 中国科学院大连化学物理研究所 | Method for constructing PRMT7 high-expression cell line and cell line |
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