CN103555734B - Coding gene of human interleukin-12, eukaryotic host cell and expression method thereof - Google Patents
Coding gene of human interleukin-12, eukaryotic host cell and expression method thereof Download PDFInfo
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Abstract
The invention provides a coding gene of human interleukin-12, a eukaryotic host cell and an expression method thereof. Specifically, the coding gene of the human interleukin-12 comprises: (a), a nucleotide sequence SEQ ID NO:9 or a complementary nucleotide sequence thereof for coding a P35 subunit of the human interleukin-12 and SEQ ID NO:10 or the complementary nucleotide sequence thereof for coding the P40 subunit of the human interleukin-12; or (b), the nucleotide sequence SEQ ID NO:21 or the complementary nucleotide sequence thereof for coding the P35 subunit of the human interleukin-12 and SEQ ID NO:22 or the complementary nucleotide sequence thereof for coding the P40 subunit of the human interleukin-12. The nucleotide sequence of the coding gene of the human interleukin-12 disclosed by the invention can enable IL-12 to stably and efficiently express in the eukaryotic host cell, especially in a Chinese hamster ovary cell (CHO cell), and to especially stably and efficiently express under a serum-free condition.
Description
The application is divisional application, and the China national application number of its original application is 201210345021.8, and the applying date is on September 17th, 2012, and denomination of invention is " encoding gene of hIL-12, eukaryotic host cell and expression method ".
Technical field
The invention belongs to technical field of bioengineering, relate to the encoding gene of hIL-12 (IL-12), eukaryotic host cell and expression method, specifically, the present invention relates to and the encoding gene of hIL-12 is optimized, thus IL-12 can be made at eukaryotic host cell, particularly in Chinese hamster ovary cell (Chinese hamster ovary celI), stability and high efficiency is expressed, and especially under serum-free condition, stability and high efficiency is expressed.
Background technology
Interleukin 12 (IL-12) is in 1989 and 1990 by discoveries such as Trinchieri and Gately, and originally called after NKSF (NKSF) and cytotoxic lymphocyte maturation factor(CLMF) (CLMF), Uniform Name in 1991 is IL-12.
Interleukin 12 is the heterodimer of a kind of 70kDa, and be made up of two covalently bound polypeptide chains, one is 35kDa(P35 subunit), another is 40kDa(P40 subunit).P35 subunit is produced by various kinds of cell, comprises T, bone-marrow-derived lymphocyte, NK cell and large mononuclear cell etc., and P40 chain produces primarily of the monocyte activated and B cell, all without the biological activity of any IL-12 during P35 and P40 Individual existence.
IL-12 has the activity regulating and participate in immune response cell, and stimulating NK cell and T cell to produce Interferon, rabbit-gamma (IFN-γ), promote the differentiation of Th1 cell, is the core factor connecting inherent immunity and acquired immunity.Meanwhile,
IL-12 can promote the recovery of hemopoietic function, the disease that the reduction for body hemopoietic function causes, as acute radiation sickness, radiation treatment, chemotherapy etc.In addition, IL-12 also has antitumor, and antiviral effect has good potential applicability in clinical practice.
Produce in the method for rIL-12 at the eukaryotic cell expression that utilizes reported at present, the expression intensity of rIL-12 is still unsatisfactory.
Culture condition and the production peak of prior art correlation technique is listed with following table 1.
The culture condition of table 1 prior art and production peak
If improve the expression amount of rhIL-12, the industrialization of rhIL-12 can be promoted.
But prior art is all that how insertion vector can promote that expression is explored, not from the viewpoint of the optimization of gene order to P35, P40.The host cell ignoring current medical protein drug is the cell in inhuman source, as the cell in mouse source, and such as the most widely used Chinese hamster ovary cell (CHO, Chinese Hamster OvaryCells).The sequence of people source IL-12 may not necessarily be best the protein expression system utilizing mouse source cell.Not from the viewpoint of the optimization of gene order.
Although the method optimizing the gene order of albumen to be expressed according to host cell has been reported, such as, the open 102268407A of Chinese patent application that name is called " a kind of HCSIF gene order and the intestinal bacteria containing this gene order ", but it provides a kind of energy high expression in the human interleukin-10 gene sequenc of microorganism.Still nobody attempts the sequence optimizing HIL-12 at present, makes it to be suitable at eukaryotic cell, the expression particularly in Chinese hamster ovary celI, the expression especially under serum-free condition.
Summary of the invention
The object of the present invention is to provide a kind of encoding gene of hIL-12, it, by the gene order optimization of people IL-12 being formed, is adapted at eukaryotic cell, particularly expresses in mouse source cell (as Chinese hamster ovary celI), thus raising expression efficiency, increase the output of albumen.
The encoding gene of hIL-12 of the present invention, comprises
(a). the SEQ ID NO:10 of the nucleotide sequence SEQ ID NO:9 of the P35 subunit of encodes human interleukin-12 or the P40 subunit of its complementary nucleotide sequence and encodes human interleukin-12 or its complementary nucleotide sequence; Or
(b). the SEQ ID NO:22 of the nucleotide sequence SEQ ID NO:21 of the P35 subunit of encodes human interleukin-12 or the P40 subunit of its complementary nucleotide sequence and encodes human interleukin-12 or its complementary nucleotide sequence.
Another object of the present invention is to provide a kind of eukaryotic host cell of expressing hIL-12, and it comprises
(a). there is the nucleotide sequence SEQ ID NO:9 of coding P35 subunit or the first expression vector of its complementary nucleotide sequence and there is the nucleotide sequence SEQ ID NO:10 of coding P40 subunit or the second expression vector of its complementary nucleotide sequence; Or
(a). there is the nucleotide sequence SEQ ID NO:21 of coding P35 subunit or the first expression vector of its complementary nucleotide sequence and there is the nucleotide sequence SEQ ID NO:22 of coding P40 subunit or the second expression vector of its complementary nucleotide sequence.
Another object of the present invention is the expression method providing a kind of hIL-12, it is characterized in that, described method comprises
(a). build respectively and comprise the nucleotide sequence SEQ ID NO:9 of coding P35 subunit or the pGN-35 plasmid of its complementary nucleotide sequence and comprise the nucleotide sequence SEQ ID NO:10 of coding P40 subunit or the pCDNA3.1 plasmid of its complementary nucleotide sequence; Or
B () builds respectively and comprises the nucleotide sequence SEQ ID NO:21 of coding P35 subunit or the pGN-35 plasmid of its complementary nucleotide sequence and comprise the nucleotide sequence SEQ ID NO:22 of coding P40 subunit or the pCDNA3.1 plasmid of its complementary nucleotide sequence
Then use the pGN-35 plasmid in step (a) or (b) and pCDNA3.1 plasmid co-transfection eukaryotic host cell, be preferably Chinese hamster ovary (CHO) cell, be more preferably CHO-DG44 or CHO-3E7 cell.
P35 and the p40 subunit that IL-12 contains is after optimizing, and can obtain the expression of stability and high efficiency in Chinese hamster ovary celI, the present inventor proves, the gene-code of p35 and the p40 subunit of people IL-12 is all high than the gene expression amount of natural people IL-12 after optimizing.Utilize the sequence of the present invention (as 3# sequence) optimized, we have obtained the stable cell line of high expression level, and in the substratum of serum-free, stable cell line can reach 200mg/L, far away higher than existing report.
Accompanying drawing explanation
Fig. 1 shows the structure collection of illustrative plates of expression vector pGN-35 and pCDNA3.1.
Fig. 2 shows transient expression assessment WB detected result (supernatant liquor).Wherein A: 72 hours CHO-3E7 cell conditioned mediums after transfection, non reducing conditions; B: 72 hours CHO-3E7 cell conditioned mediums after transfection, reductive condition; C: 72 hours CHO-DG44 cell conditioned mediums after transfection, non reducing conditions; D: 72 hours CHO-DG44 cell conditioned mediums after transfection, reductive condition;
Abbreviation: P:IL-12 albumen, as positive control (Genscript, product article No. Z02709); NC: the substratum supernatant of non-transfected cells, as negative control; M: mark (Marker); 1,2,3 ... 9,10: distinguish the transfection cell conditioned medium liquid of 1 to No. 10 alternative plasmid;
Fig. 3 shows transient expression assessment WB detected result (cell pyrolysis liquid).Wherein A: 72 hours CHO-3E7 lysis liquid after transfection, non reducing conditions; B: 72 hours CHO-DG44 cell conditioned mediums after transfection, non reducing conditions;
Abbreviation: NC: the cell pyrolysis liquid of non-transfected cells, as negative control; M: mark (Marker); 1,2,3 ... 9,10: distinguish the transfection cell pyrolysis liquid of 1 to No. 10 alternative plasmid
Fig. 4 shows Western-blot detected result (non reducing conditions).Wherein NC: the substratum supernatant of non-transfected cells, as negative control; Native, #3, #9: distinguish transfection native gene sequence, the cell conditioned medium of the alternative gene order of #3 and No. 9 alternative gene orders;
Note: the primary antibodie of use is anti-p40 antibody (IL-12antibody(p40), AbcamTM, article No.: ab91216).
Embodiment
The present invention is described in detail below.
Interleukin 12 (IL-12) is the heterodimer of a kind of 70kDa, and be made up of two covalently bound polypeptide chains, one is 35kDa(P35 subunit), another is 40kDa(P40 subunit).The natural acid sequence of IL-12 is in table 2.
Table 2 IL-12 aminoacid sequence (containing natural signals peptide)
The present inventor's trial mouse IgG signal peptide replaces natural signals peptide to improve the expression (table 3) of IL-12, and use OptimumGeneTM software to be optimized the amino acid whose DNA sequence dna of coding IL-12 respectively for natural signals peptide and mouse IgG signal peptide, the gene order optimized utilizes different signal peptides (natural signals peptide, or mouse source signal peptide) to amount to formation 10 groups of alternative sequence (table 4).
Wherein, 1-5 alternative sequence carrys out the IL-12 recombinant protein aminoacid sequence of self-contained natural signals peptide; 6-10 alternative sequence carrys out the IL-12 recombinant protein aminoacid sequence of self-contained mouse IgG signal peptide.
Table 3 IL-12 aminoacid sequence (containing mouse IgG signal peptide)
10 groups of alternative DNA sequences after table 4 optimization
Through experiment, the present inventor find the alternative sequence 3 containing natural signals peptide in table 4 and the expression amount of alternative sequence 9 containing mouse source signal peptide the highest.
Therefore, one object of the present invention provides a kind of encoding gene of hIL-12, and it comprises
(a). the SEQ ID NO:10 of the nucleotide sequence SEQ ID NO:9 of the P35 subunit of encodes human interleukin-12 or the P40 subunit of its complementary nucleotide sequence and encodes human interleukin-12 or its complementary nucleotide sequence; Or
(b). the SEQ ID NO:22 of the nucleotide sequence SEQ ID NO:21 of the P35 subunit of encodes human interleukin-12 or the P40 subunit of its complementary nucleotide sequence and encodes human interleukin-12 or its complementary nucleotide sequence.
Described complementary nucleotide sequence is the nucleotide sequence with indication nucleotide sequence complete complementary.
Preferably, the encoding gene of described hIL-12 comprises the nucleotide sequence SEQ ID NO:9 of coding P35 subunit and the SEQ ID NO:10 of coding P40 subunit; Or comprise the nucleotide sequence SEQ ID NO:21 of coding P35 subunit and the SEQ ID NO:22 of coding P40 subunit.
Another object of the present invention is to provide the eukaryotic host cell of expressing hIL-12, and it comprises
(a). there is the nucleotide sequence SEQ ID NO:9 of coding P35 subunit or the first expression vector of its complementary nucleotide sequence and there is the nucleotide sequence SEQ ID NO:10 of coding P40 subunit or the second expression vector of its complementary nucleotide sequence; Or
(a). there is the nucleotide sequence SEQ ID NO:21 of coding P35 subunit or the first expression vector of its complementary nucleotide sequence and there is the nucleotide sequence SEQ ID NO:22 of coding P40 subunit or the second expression vector of its complementary nucleotide sequence.
In the present invention, above-mentioned eukaryotic host cell is preferably the cell in the inhuman source that current medical protein drug field is commonly used, as non-human mammalian cell line.The breeding of mammalian cell in culture is well known in the art.Preferably mammalian cell is many commercially available clones, include but not limited to, Chinese hamster ovary cell (CHO, Chinese Hamster Ovary Cells), Vero cell, HeLa cell, young hamster kidney (BHK) cell, monkey-kidney cells (COS) etc.The particularly preferred cell of the present invention is the cell in mouse source, and such as the most widely used Chinese hamster ovary cell, is more preferably CHO-DG44 or CHO-3E7 cell.
Expression vector typically refers to the plasmid or other nucleic acid molecule that can copy in selected host cell.This expression vector can self-replicating, or by copying in the genome of method Insertion Into Host Cell well known in the art.
In an example of the present invention, first expression vector with the nucleotide sequence of coding P35 subunit is pGN-35 plasmid, and second expression vector with the nucleotide sequence of coding P40 subunit is pCDNA3.1 plasmid.
Present invention employs dual-expression vector, its advantage is, the plasmid at two subunit places contains different screening-genes, for example, the plasmid at the subunit place of coding P35 can contain DHFR gene, can pressurize to increase with MTX in CHO-DG44 cell and screen the cell of P35 expression, and the plasmid at the subunit place of the P40 that encodes is containing Neomycin gene, can screen the cell of P40 expression with Neomycin.If cell expresses P35 and P40 two subunits, then under surviving in the condition of Double Selection simultaneously.Those skilled in the art also can adopt other expression vectors well-known to those skilled in the art and screening-gene.
Another object of the present invention additionally provides the expression method of hIL-12, and it comprises
(a). build respectively and comprise the nucleotide sequence SEQ ID NO:9 of coding P35 subunit or the pGN-35 plasmid of its complementary nucleotide sequence and comprise the nucleotide sequence SEQ ID NO:10 of coding P40 subunit or the pCDNA3.1 plasmid of its complementary nucleotide sequence; Or
B () builds respectively and comprises the nucleotide sequence SEQ ID NO:21 of coding P35 subunit or the pGN-35 plasmid of its complementary nucleotide sequence and comprise the nucleotide sequence SEQ ID NO:22 of coding P40 subunit or the pCDNA3.1 plasmid of its complementary nucleotide sequence
Then use the pGN-35 plasmid in step (a) or (b) and pCDNA3.1 plasmid co-transfection eukaryotic host cell, be preferably Chinese hamster ovary (CHO) cell, be more preferably CHO-DG44 or CHO-3E7 cell.
Transfection refers to and is imported in host cell by the expression vector containing interested nucleic acid.Transfection method generally includes: electroporation; Adopt the transfection of calcium chloride, DEAE-dextran or other materials; Microparticle bombardment; Liposome transfection; Infect and additive method (see " the Molecular Cloning: A Laboratory guide " the 2nd edition of the people such as Sambrook, 1989 years).In an example of the present invention, transfection method is electroporation or liposome transfection.
The cotransfection eukaryotic host cell obtained especially is applicable to being incubated in the substratum of serum-free.
In the present invention, p35 and the p40 subunit that IL-12 contains, after optimizing, at eukaryotic cell, particularly can obtain the expression of stability and high efficiency in Chinese hamster ovary celI, all high than the gene expression amount of natural people IL-12.Utilize the sequence of the present invention optimized, we have obtained the stable cell line of high expression level, and in the substratum of serum-free, stable cell line can reach 200mg/L, far away higher than existing report.
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
The choning and sequencing of p35 and the p40 subunit gene sequence that embodiment 1IL-12 contains
Use P35 and the P40 natural acid sequence in UniProt database, according to kind of the alternative gene of 10 in conceptual design, replace some codon in sequence.Through gene sequencing, its result is consistent with expected sequence in table 4.
The structure of embodiment 2 plasmid and mensuration
The gene order of 10 groups of p35 and the p40 subunits optimized in table 4 is cloned into pGN-35 and pCDNA3.1(Fig. 1 respectively) expression plasmid.Consider that the expression amount of p35 subunit is often lower than p40 subunit, the p35 subunit of often pair of gene utilizes EcoR1 and Xba1 restriction enzyme site to be cloned into pGN-35 plasmid, so that the function of the gene amplification utilizing DHFR gene adjoint when MTX pressurization is cultivated, the gene of amplification p35 subunit.The sequence of the p40 subunit of often pair of gene utilizes EcoR1 and Xba1 restriction enzyme site to be cloned into pCDNA3.1 plasmid, utilizes Neomycin drug resistant gene to select the cell of p40 subunit expression.Sequential analysis is carried out, to confirm the exactness of sequence after clone.
The expression of embodiment 3rhIL-12 albumen
Transient expression is carried out, to assess the effect of majorizing sequence containing p35 and containing p40 subunit expression plasmids by what obtain.In transient expression evaluation test, have chosen CHO-DG44 and CHO-3E7 two strain host cell.Wherein, CHO-DG44 be build industrial production level stable cell line the host cell commonly use, screen the stable cell lines that can obtain high yield through pressurization, but when for transient expression, its expression amount is often not high.Thus, test employs transient expression host cell CHO-3E7 simultaneously and is used as alternative reference, to make up the deficiency of CHO-DG44 in transient expression assessment.
In transient expression evaluation test, by 10 groups of alternative plasmids respectively transfection enter CHO-DG44 cell and CHO-3E7 cell.Concrete operations are as follows: CHO-DG44 and CHO-3E7 cell is cultivated at Opti-CHO serum free medium (Gibco, Cat#12681) respectively.Wherein by electroporation, transfection is carried out to CHO-DG44 cell, use L-PEI (PEI) to carry out transfection to CHO-3E7 cell.Whether the engineering cell after transfection, after the short period of time cultivates, measures IL-12 in supernatant liquor by Western-blot and ELISA two kinds of methods and expresses and transient expression amount.By comparing IL-12 transient expression amount, picking out DNA sequence dna and the expression vector of the relatively high IL-12 of Transient Expression amount, setting up with stable cell line later.
Wherein, CHO-3E7 cell Polyethylenimine ' ' MAX ' ' linear, MW25kDa (40kDanominal), 3mg/mL, pH7.0 (Polysciences Inc.cat#24765-2). transfection is before 24 hours, by CHO-3E7 cell dilution to 0.5x10
6cells/mL.Cell concn during transfection should be 1.5to2.0x10
6cells/mL.The cell separating 17ml, to new culturing bottle, by 1.47mL transfection media (FreeStyleTMCHO medium) 37 DEG C of preheatings, adds 30 μ g DNA plasmids (30 μ L), slightly shakes mixing.90 μ L LPEI MAX solution and 1.41mL transfection liquid mix, and cumulative volume is 1.5mL.By 1.5mL LPEIMAX solution with slightly shake mixing 4 second containing the transfection liquid of plasmid, ambient temperatare puts 8-10 minute.Then, added in 17mL cell by this 3mL mixed solution, fully mix with cell, the horizontal shaker putting what 120rpm cultivates the method for (5%CO2,37 DEG C) .CHO-DG44 cell electrotransfection.In CD OptiCHO substratum, 10x10
7cell is placed in electrotransfection pipe, by 40 μ g plasmid DNA and 100 μ l ddH
2o mixes.The surge voltage of Gene PulserXcell electroporation apparatus is located at 300V.(40 microgram) plasmid of 100 microlitres is added, 700 microlitres of cells (10 in electrotransfection pipe
7cells), cumulative volume 800 microlitre.Cell is transferred to 10 milliliters of culture dish immediately after electrotransfection.
Above cell is all cultivated at 37 degrees Celsius, 5%CO2 cell culture incubator.Collect supernatant after 72 hours and carry out ELISA(in table 5) and Western Blot(see Fig. 2, A, B, C, D) detect, collect cell simultaneously, carry out Western Blot(after cracking and see A, B in Fig. 3) detect.
Table 5 transient expression assessment ELISA detected result
Above-mentioned ELISA and WB result display, IL-12 has expression in above-mentioned two CHO expression systems.Higher alternative group of ELISA detected result signal in WB detected result is also relatively strong, shows that ELISA with WB detected result is consistent substantially.Containing the alternative sequence (No. 3 and No. 4) of natural signals peptide, in CHO-DG44 host cell, obtain higher expression; And in CHO-3E7 host cell, obtain higher expression containing the alternative sequence (No. 9 and No. 7) of mouse source signal peptide.
According to above result, consider the expression effect of two cell strains, containing No. 3, the alternative sequence of natural signals peptide and the highest containing the expression amount of No. 9 of mouse source signal peptide.
Comparing of the people IL-12 gene order expressing quantity of embodiment 4 natural human IL-12 gene order and optimization
The native gene sequence (table six) of p35 and the p40 subunit of people IL-12 is cloned into pGN-35 and pCDNA3.1 respectively, and order-checking ensures the correct of sequence.
Table 6 natural IL-12 recombinant protein DNA sequence dna
In transient expression evaluation test, by natural IL-12 gene order, No. 3 alternative gene orders and No. 9 alternative gene orders respectively transfection enter CHO-DG44 cell and CHO-3E7 cell, the condition of cell cultures and the method for plasmid transfection are with embodiment 3.Collect supernatant after 72 hours and carry out ELISA(table seven) and Western Blot detection (Fig. 4).
| CHO-DG44 cell | CHO-3E7 cell | |
| Native gene sequence | 163.86 | 308.85 |
| No. 3 alternative gene orders | 303.65 | 1404.87 |
| No. 9 alternative gene orders | 588.70 | 3276.57 |
Table 7 ELISA detected result (μ g/L)
Above-mentioned Western-blot and ELISA result display, IL-12 native gene sequence has expression in CHO-DG44 and CHO-3E7 two host cells.But in two expression systems, the expression amount of native gene sequence is all minimum, be secondly No. 3 alternative gene orders, and No. 9 alternative gene orders is the highest.
No. 3, embodiment 5.DNA sequence can have the expression of the people's rIL-12 more than 200mg/L at stable cell line
By electroporation, No. 3 sequence pair CHO-DG44 cells are carried out transfection.After 72 hours, the density of the cell after transfection by 200 cells in every hole is inoculated in 96 porocyte culture dish.The MTX adding G418 and 50nM of 200 μ g/ml in the substratum of cell screens cell.Cell Dot Blot and ELISA of existence is screened.The pressurization screening of MTX is from 50nM, 200nM to 500nM.Positive colony after 500nM pressurization screening carries out subclone.Under 200nM to 500nM pressurization level, part clone cell expressing the results are shown in ELISA result table eight.By the data in table eight, the stabilized cell clone obtained according to No. 3 sequences has and multiplely reaches more than 200mg/L.Confirm, the report that the sequence after optimization is expressed in the existing native sequences of the expression ratio of Chinese hamster ovary celI is more superior.The expression of results of this stabilized cell clone also confirms, the result that different sequence is cloned at height and the stabilized cell of transient transfection expression amount is identical.Namely the expression amount of the sequence that expression amount is high when transient expression after stabilized cell colony screening is also high.
After the screening of table eight subclone, part cell clone ELISA detected result
| Clone number | Expression amount (mg/L) | Clone number | Expression amount (mg/L) |
| 41 | 170.02 | 163 | 129.97 |
| 52 | 120.35 | 165 | 94.70 |
| 61 | 180.37 | 170 | 72.55 |
| 79 | 98.15 | 344 | 76.55 |
| 83 | 71.56 | 640 | 185.00 |
| 84 | 137.42 | 652 | 210.69 |
| 135 | 118.53 | 717 | 108.91 |
| 141 | 113.06 | 763 | 102.07 |
| 151 | 167.22 | 773 | 135.95 |
| 158 | 145.89 | 842 | 100.28 |
| NC | R |
After the screening of table eight subclone, part cell clone ELISA detected result
Embodiment 6 natural human IL-12 gene order compares with optimization descendant's IL-12 gene order
The p35 gene order of 1 natural human IL-12 with optimize after the Gene sequence comparison (up is native sequences, and descending is the p35 subunit sequence of 3# sequence after optimizing) of p35 subunit in 3# sequence
.2 the p40 gene order of natural human IL-12 and the Gene sequence comparison of the p40 subunit optimized in rear 3# sequence
(up is native sequences, and descending is the p40 subunit sequence of 3# sequence after optimizing)
3. natural human IL-12 p35 gene order with optimize after the Gene sequence comparison (up is native sequences, and descending is the p35 subunit sequence of 9# sequence after optimizing, is the sequence of mouse IgG signal peptide before the yellow password indicated) of p35 subunit in 9# sequence
4. natural human IL-12 p40 gene order with optimize after the Gene sequence comparison (up is native sequences, and descending is the p40 subunit sequence of 9# sequence after optimizing, is the sequence of mouse IgG signal peptide before the yellow password indicated) of p40 subunit in 9# sequence
P35 and the p40 subunit that IL-12 contains, after optimizing, can obtain the expression of stability and high efficiency in Chinese hamster ovary celI, and No. 3 optimized gene sequences (containing natural signals peptide) are after expressing, and in CHO-DG44 cell, expression amount is 2 times of natural gene; No. 9 optimized gene sequences (containing mouse source signal peptide) are after expressing, and expression amount is 3 times of natural gene.In CHO-3E7 cell, No. 3 optimized gene sequence expression amounts are 4 times of natural gene; No. 9 optimized gene sequences (containing mouse source signal peptide) are after expressing, and expression amount is 10.6 times of natural gene.These digital proofs, the gene-code of p35 and the p40 subunit of people IL-12 is all high than the gene expression amount of natural people IL-12 after optimizing.Utilize the 3# sequence optimized, we have obtained the stable cell line of high expression level, in the substratum of serum-free, can reach 200mg/L far away higher than existing report.
Claims (11)
1. the encoding gene of hIL-12, is characterized in that, described encoding gene is made up of following sequence:
The SEQ ID NO:22 of the nucleotide sequence SEQ ID NO:21 of the P35 subunit of encodes human interleukin-12 or the P40 subunit of its complete complementary nucleotide sequence and encodes human interleukin-12 or its complete complementary nucleotide sequence.
2. encoding gene as claimed in claim 1, is characterized in that, described encoding gene is made up of the nucleotide sequence SEQ ID NO:21 of P35 subunit of encoding and the SEQ ID NO:22 of coding P40 subunit.
3. express an eukaryotic host cell for hIL-12, it is characterized in that, described eukaryotic host cell comprises:
There is the nucleotide sequence SEQ ID NO:21 of coding P35 subunit or the first expression vector of its complete complementary nucleotide sequence and there is the nucleotide sequence SEQ ID NO:22 of coding P40 subunit or the second expression vector of its complete complementary nucleotide sequence.
4. eukaryotic host cell as claimed in claim 3, it is characterized in that, described eukaryotic host cell comprises first expression vector of the nucleotide sequence SEQ ID NO:21 with coding P35 subunit and has second expression vector of SEQ ID NO:22 of coding P40 subunit.
5. the eukaryotic host cell as described in claim 3 or 4, is characterized in that, described eukaryotic host cell is Chinese hamster ovary (CHO) cell.
6. the eukaryotic host cell as described in claim 3 or 4, is characterized in that, described eukaryotic host cell is CHO-DG44 or CHO-3E7 cell.
7. the eukaryotic host cell as described in claim 3 or 4, is characterized in that, described first expression vector is pGN-35 plasmid.
8. the eukaryotic host cell as described in claim 3 or 4, is characterized in that, described second expression vector is pCDNA3.1 plasmid.
9. an expression method for hIL-12, is characterized in that, described method comprises:
Build respectively and comprise the nucleotide sequence SEQ ID NO:21 of coding P35 subunit or the pGN-35 plasmid of its complete complementary nucleotide sequence and comprise the nucleotide sequence SEQ ID NO:22 of coding P40 subunit or the pCDNA3.1 plasmid of its complete complementary nucleotide sequence;
Then with described pGN-35 plasmid and described pCDNA3.1 plasmid co-transfection eukaryotic host cell.
10. expression method as claimed in claim 9, it is characterized in that, described eukaryotic host cell is Chinese hamster ovary (CHO) cell.
11. expression methods as claimed in claim 9, it is characterized in that, described eukaryotic host cell is CHO-DG44 or CHO-3E7 cell.
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| CN106636123B (en) * | 2015-10-15 | 2020-06-30 | 广东科玮生物技术股份有限公司 | Method for constructing rhIL-12 expression system in CHO cell |
| ES2941411T3 (en) * | 2016-05-18 | 2023-05-22 | Modernatx Inc | Polynucleotides encoding interleukin-12 (IL12) and uses thereof |
| CN108251375B (en) * | 2016-12-28 | 2021-05-07 | 康立泰药业有限公司 | Method for producing recombinant human interleukin-12 by fermentation |
| CN109486828B (en) * | 2018-12-27 | 2022-05-31 | 广东暨大基因药物工程研究中心有限公司 | Gene for coding recombinant human interleukin 12 and application thereof |
| US20230000969A1 (en) * | 2019-10-24 | 2023-01-05 | Inovio Pharmaceuticals, Inc. | Improved vaccines for recurrent respiratory papillomatosis and methods for using the same |
| CN114395041B (en) * | 2021-02-09 | 2023-12-01 | 甘李药业股份有限公司 | Method for preparing anti-IL-12 and/or IL-23 monoclonal antibody |
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| CN100518817C (en) * | 2003-07-04 | 2009-07-29 | 南加州大学 | Therapeutic agent to recover hematopoiesis function and its compositions and usage |
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| CN1467292A (en) * | 2003-05-27 | 2004-01-14 | 中国科学技术大学 | Highly effective expressing method of interleukin-12 |
| CN101058807A (en) * | 2006-05-26 | 2007-10-24 | 神州细胞工程有限公司 | Optimized monoclonal antibody |
| CN101200730A (en) * | 2007-07-02 | 2008-06-18 | 广州市恺泰生物科技有限公司 | IL-12 expression carrier as well as eukaryotic cell strain expressed thereby and uses thereof |
| CN101638657A (en) * | 2008-12-17 | 2010-02-03 | 张文卿 | Construction and application of recombinant human interleukin 12 eukaryotic expression vector |
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