CN111471655B - Anti-human IL12/23 stable transgenic cell strain and construction method and application thereof - Google Patents
Anti-human IL12/23 stable transgenic cell strain and construction method and application thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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Abstract
The invention discloses an anti-human IL12/23 stable transgenic cell strain, a construction method and application thereof, wherein the cell strain is named as an anti-human IL12/23CHO-S stable transgenic cell strain, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC No. C202050) at the university of Wuhan in 3 months and 11 days in 2020. The yield of the anti-human IL12/23 antibody of the anti-human IL12/23 stable transgenic cell strain CHO-S can reach 2.9g/L, which is 6 times of that of the existing anti-human IL12/23 antibody expression cell strain; the antibody yield is stable and the antibody quality is high between different production batches.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to an anti-human IL12/23 stable transgenic cell strain, a construction method and application thereof.
Background
IL12/23 (interleukin 12/interleukin 23) is a cytokine that naturally occurs in inflammation and immune responses and is thought to play a key role in immune-mediated inflammatory diseases, including plaque psoriasis, psoriatic arthritis, crohn's disease, systemic lupus erythematosus, and the like.
Anti-human IL12/23 monoclonal antibodies can inhibit 2 pro-inflammatory cytokines by binding to the p40 subunit shared by IL-12 and IL-23 and preventing the binding to the receptor IL-12β1 on the cell surface, so as to reduce inflammatory response caused by overproduction and release of IL12/23, and have important clinical significance for treating diseases caused by overproduction of IL 12/23. Anti-human IL12/23 mab (Wu Sinu mab) is a self-injectable biologic therapeutic that was approved by the national food and drug administration (CFDA) for the treatment of psoriasis for 1 indication.
The traditional preparation method of the monoclonal antibody secreting cell strain is a fusion method of immune mouse spleen cells and myeloma cells, and comprises the processes of antigen immunization, cell fusion, cell strain screening, cell bank construction and the like. Since the mouse antibody is a foreign antigen with respect to the human body, the mouse monoclonal antibody causes serious rejection reaction in the human body after being injected into the human body. Meanwhile, the fused cell strain prepared by the traditional method is easy to generate the phenomena of chromosome loss, gene deletion and the like, so that the stability among different batches in the monoclonal antibody production process is seriously influenced, and the product quality is influenced. In addition, the fusion cell line has the problems of low antibody yield, difficult cell culture and the like.
Disclosure of Invention
The invention aims to provide an anti-human IL12/23 stable transgenic cell line with higher anti-human IL12/23 antibody yield, and a construction method and application thereof.
In order to achieve the above object, the technical scheme of the present application is as follows:
an anti-human IL12/23 stable transgenic cell line, which is named as an anti-human IL12/23CHO-S stable transgenic cell line, is preserved in China center for type culture Collection, with the preservation number of CCTCC No. C202050, which is located at university of Wuhan, at 11/3/2020.
The construction method of the anti-human IL12/23 stable transgenic cell strain comprises the following steps:
(1) Preparing plasmids containing heavy chain genes of the anti-human IL12/23 antibody and plasmids containing light chain genes of the anti-human IL12/23 antibody, and co-transfecting the plasmids into parent CHO-S cells;
(2) Puromycin/G418 screening is carried out on the CHO-S cells after transfection to obtain stable transgenic cells;
specifically, the Puromycin/G418 screening comprises: culturing the transfected cells in a complete culture solution, replacing the complete culture solution with a screening culture solution containing 10ug/mL puromycin and 800ug/mL G418 after 48 and h, and replacing the screening culture solution once every 3-4 days until the cells grow stably;
preferably, the final concentration of puromycin and G418 in the screening medium is 10ug/mL and 800ug/mL, respectively.
Preferably, the Puromycin/G418 screening further comprises: and inoculating the cells which grow stably in the screening culture solution into the complete culture solution for continuous culture to obtain the successfully transfected stably transfected cells.
Preferably, the cells grown stably in the selection medium are inoculated into the complete medium for continuous culture for 2-3 days, and after inoculation, the cell density in the complete medium is 5X 10 5 cells/mL。
(3) And (3) carrying out monoclonal cell strain sorting on the stable transfer cells to obtain the anti-human IL12/23 stable transfer cell strain CHO-S.
Specifically, the monoclonal cell strain sorting comprises: and inoculating the monoclonal cell strains in the stable transfer cells into a complete culture solution one by one, continuously culturing for 12-14 days, and detecting the yield of the anti-human IL12/23 antibody of each monoclonal cell strain to obtain the anti-human IL12/23 stable transfer cell strain CHO-S.
The invention also provides an application of the anti-human IL12/23 stable transgenic cell strain in preparing an anti-human IL12/23 antibody, which comprises the following steps:
(a) Inoculating the anti-human IL12/23 stable transfer cell strain CHO-S into shake flask containing complete CD culture solution, placing at 37deg.C, 5% CO 2 Subculturing at 120 rpm;
(b) After subculturing, transferring the cells into a large-volume culture shake flask, and inoculating cells with initial density of more than 3×10 5 cells/mL; in order to make the culture solution have higher dissolved oxygen, the volume of the culture solution in the culture shake flask is not more than 1/5 of the total volume of the culture flask;
(c) When the cell density reaches 1X 10 6 cutting off the supply of carbon dioxide after cell/mL, and regulating the rotating speed of the shaking table to 130-135 rpm;
(d) Culturing for 6-8 days, collecting culture solution, centrifuging, filtering with 0.45 μm filter membrane to obtain cell culture supernatant, and purifying and eluting cell culture supernatant sequentially to obtain anti-human IL12/23 antibody.
Preferably, in step (a), the cells are passaged at least 2 times, and the cell density in the culture solution during the subculture is not more than 2X 10 6 cells/mL。
The yield of the anti-human IL12/23 antibody of the anti-human IL12/23 stable transgenic cell strain CHO-S can reach 3g/L, which is 6 times of that of the existing anti-human IL12/23 antibody expression cell strain.
Compared with the prior art, the invention has the beneficial effects that:
(1) The yield of the anti-human IL12/23 antibody of the anti-human IL12/23 stable transgenic cell strain CHO-S can reach 3g/L, which is 6 times of that of the existing anti-human IL12/23 antibody expression cell strain; the antibody yield is stable and the antibody quality is high between different production batches.
(2) The construction method of the anti-human IL12/23 stable transgenic cell line has simple and convenient steps and is easy to implement.
Drawings
FIG. 1 is an elution profile of the GE AKTA Pure protein isolation and purification system eluting the purified cell culture supernatant.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed description.
Example 1
The embodiment relates to a construction method of an anti-human IL12/23 stable transgenic cell strain, which comprises the following steps:
(1) Preparing plasmids containing heavy chain genes of the anti-human IL12/23 antibody and plasmids containing light chain genes of the anti-human IL12/23 antibody, and co-transfecting the plasmids into parent CHO-S cells;
the nucleotide sequence of the heavy chain gene of the anti-human IL12/23 antibody is shown as SEQ ID No.1, the nucleotide sequence of the light chain gene of the anti-human IL12/23 antibody is shown as SEQ ID No.2, and the preparation method of the plasmid is carried out according to the description of a QIAGEN kit; the plasmid transfection procedure was performed according to the Invitrogen kit.
The heavy chain gene nucleotide sequence SEQ ID No.1 of the anti-human IL12/23 antibody is as follows:
GAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCAGGAGAGAGCCTG AAGATCAGCTGTAAGGGGAGCGGATATAGTTTCACAACATATTGGCTGGGATGGGTGAGGCAGATGCCCGGAAAAGGACTGGACTGGATCGGAATTATGTCACCCGTGGACAGCG ACATCAGATACAGCCCATCCTTTCAGGGGCAGGTGACCATGAGCGTGGATAAGAGCATCACAACAGCCTACCTGCAGTGGAACAGCCTGAAGGCCAGCGACACCGCCATGTACTAC TGCGCCAGAAGGAGACCCGGCCAGGGGTACTTCGATTTCTGGGGCCAGGGAACACTGGTGACAGTGTCTTCCAGCAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA GGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGACGAGCTGACCAAGAACCAGGTCAGC CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC CTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAATGA
the anti-human IL12/23 antibody light chain gene nucleotide sequence SEQ ID No.2 is:
GACATTCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGA GTGACCATCACCTGCAGAGCCAGCCAGGGCATCTCCAGCTGGCTGGCCTGGTACCAGCAGAAACCCGAGAAGGCCCCAAAAAGCCTGATCTACGCCGCCAGTAGCCTGCAGAGCG GAGTGCCCAGCAGATTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAG CAGCCTGCAGCCCGAAGACTTCGCCACCTACTACTGCCAGCAGTACAACATCTACCCCT ACACCTTCGGCCAGGGCACCAAACTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCC TGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTA CAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGG GGAGAGTGTTGA
(2) Puromycin/G418 screening is carried out on the CHO-S cells after transfection to obtain stable transgenic cells;
the method specifically comprises the following steps:
(2.1) culturing the transfected cells in a complete culture solution, changing the complete culture solution to a screening culture solution containing 10ug/mL puromycin and 800ug/mL G418 after 48 hours, and changing the screening culture solution once every 3-4 days until the cells grow steadily;
(2.2) will be in the screening MediumThe cells with stable growth are inoculated into a complete culture solution, and after inoculation, the cell density in the complete culture solution is 5 multiplied by 10 5 cells/mL; after 2-3 days of continuous culture, the anti-human IL12/23 antibody yield of the cells is detected to determine whether the plasmid is successfully transfected or not and whether the Puromycin/G418 screening is successful or not, and finally stable transgenic cells (polyclonal) are obtained;
(3) And (3) carrying out monoclonal cell strain sorting on the stable transfer cells to obtain an anti-human IL12/23 stable transfer cell strain CHO-S.
The method specifically comprises the following steps:
(3.1) inoculating stable-rotation cells into a 96-well plate after multiple dilutions, wherein each well contains 200 mu L of complete culture solution, and ensuring that each well contains only one stable-rotation monoclonal cell strain;
(3.2) culturing the cells in the 96-well plate for about 7 days, observing the growth state of the cells by using a microscope frequently, and marking the culture holes of each monoclonal cell strain;
(3.3) detecting the yield of the anti-human IL12/23 antibody of each monoclonal cell strain after continuously culturing for 12-14 days, selecting a high-yield monoclonal cell strain to transfer into a 24-well plate, continuously culturing for 12-14 days, and detecting the yield of the anti-human IL12/23 antibody of the monoclonal cell strain in each well; continuously selecting a high-yield monoclonal cell strain from a 24-well plate, transferring the monoclonal cell strain into a 6-well plate, continuously culturing for 12-14 days, detecting the yield of the anti-human IL12/23 antibody of the monoclonal cell strain in each well, and selecting a monoclonal cell strain with the highest yield of the anti-human IL12/23 antibody from the monoclonal cell strain, namely the anti-human IL12/23 stable-transfer cell strain CHO-S of the embodiment;
(3.4) the anti-human IL12/23 stable transgenic cell strain CHO-S is frozen and maintained.
As can be seen from the result of PAGE gel electrophoresis of cell culture supernatant of the anti-human IL12/23 stable cell strain CHO-S and standard antibody protein IgG (shown in FIG. 1), the content of the anti-human IL12/23 antibody in the cell culture supernatant of the anti-human IL12/23 stable cell strain CHO-S is higher than the content of 160 mug/mL of IgG.
Example 2
This example uses the anti-human IL12/23 stable cell line CHO-S obtained in example 1 to produce anti-human IL12/23 antibodies, which comprises the steps of:
(1) (a) transfer of anti-human IL12/23 stable cell line CHO-S (hereinafter referred to as cell line CHO-S) into a 125mL shake flask containing 30mL of complete CD culture solution, and place the shake flask at 37℃and 5% CO 2 Shake culturing in a shaking table at 120 rpm;
the cryopreserved anti-human IL12/23 stable transgenic cell strain CHO-S needs to be resuscitated in advance, and the resuscitating method comprises the following steps: taking out the cell strain CHO-S from a liquid nitrogen tank, quickly thawing in a water bath kettle at 37 ℃, centrifuging the cell liquid, removing the supernatant, and then washing with PBS;
at the initial culture, the cell strain CHO-S is subcultured every 2-3 days, at least more than 2 times, and the cell density is prevented from being too high (not more than 2×10) 6 cell/mL)；
(b) After subculturing, transferring the cells into a large-volume culture shake flask, and inoculating cells with initial density of more than 3×10 5 cells/mL; in order to make the culture solution have higher dissolved oxygen, the volume of the culture solution in the culture shake flask is not more than 1/5 of the total volume of the culture flask;
(c) After culturing for 48 hours, the cell density in the culture solution was examined by a microscope to determine whether it reached 1X 10 6 cell/mL (should reach or be higher) until cell density reaches 1×10 6 cutting off the supply of carbon dioxide after cell/mL, and regulating the rotating speed of the shaking table to 130-135 rpm;
(d) Culturing for 6-8 days (the cell density in culture solution can reach 7-8X10) 6 cell/mL), collecting culture solution, centrifuging and filtering with 0.45 μm filter membrane to obtain cell culture supernatant, sequentially purifying and eluting cell culture supernatant by using GE AKTA Pure protein separation and purification system, and eluting with the curve shown in figure 1.
As shown in FIG. 1, the response of anti-human IL12/23 antibody in the eluate was up to approximately 200, indicating a higher concentration of anti-human IL12/23 antibody in the eluate.
A standard protein concentration detection curve is prepared, 100mL of culture solution is purified to obtain 6mL of antibody, and the antibody is diluted by four concentration gradients to monitor the protein concentration, so that the following results shown in Table 1 are obtained:
TABLE 1
The final concentration of antibody was measured as: 4.82mg/mL. The final antibody yield in 100mL of culture was 28.92mg. The antibody yield of the cell line was estimated to be 2.9g/L.
Sequencing the obtained anti-human IL12/23 antibody by the Shanghai bioscience research institute, and the sequencing result shows that the amino acid sequence of the heavy chain of the anti-human IL12/23 antibody is shown as SEQ ID No.3, and the amino acid sequence of the light chain of the anti-human IL12/23 antibody is shown as SEQ ID No. 4. The results of peptide coverage analysis by the institute of biochemistry and cell biology (Institute of Biochemistry and Cell Biology), the institute of Shanghai bioscience (Shanghai Institutes For Biological Sciences) and the national academy of sciences (Chinese Academy of Sciences) indicate that the anti-human IL12/23 antibody of the invention has 100% identity with the sequence of Wu Sinu mab.
Heavy chain amino acid sequence of anti-human IL12/23 antibody SEQ ID No.3 is EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMSPVDSDIRYSPSFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPGQGYFDFWGQGTLVT VSSSSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK
The anti-human IL12/23 antibody light chain amino acid sequence SEQ ID No.4 is:
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
sequence listing
<110> Zhejiang Zhengxi biological medicine Co., ltd
<120> anti-human IL12/23 stable transgenic cell line, construction method and application thereof
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gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc 60
atcacctgca gagccagcca gggcatctcc agctggctgg cctggtacca gcagaaaccc 120
gagaaggccc caaaaagcct gatctacgcc gccagtagcc tgcagagcgg agtgcccagc 180
agattcagcg gaagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaagacttcg ccacctacta ctgccagcag tacaacatct acccctacac cttcggccag 300
ggcaccaaac tggaaatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagttcgc ccgtcacaaa gagcttcaac aggggagagt gttgagaggt gcagctggtg 660
cagagcggag ccgaggtgaa gaagccagga gagagcctga agatcagctg taaggggagc 720
ggatatagtt tcacaacata ttggctggga tgggtgaggc agatgcccgg aaaaggactg 780
gactggatcg gaattatgtc acccgtggac agcgacatca gatacagccc atcctttcag 840
gggcaggtga ccatgagcgt ggataagagc atcacaacag cctacctgca gtggaacagc 900
ctgaaggcca gcgacaccgc catgtactac tgcgccagaa ggagacccgg ccaggggtac 960
ttcgatttct ggggccaggg aacactggtg acagtgtctt ccagcagcac caagggccca 1020
tcggtcttcc ccctggcacc ctcctccaag agcacctctg ggggcacagc ggccctgggc 1080
tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 1140
accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1200
agcgtggtga ccgtgccctc cagcagcttg ggcacccaga cctacatctg caacgtgaat 1260
cacaagccca gcaacaccaa ggtggacaag agagttgagc ccaaatcttg tgacaaaact 1320
cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 1380
cccccaaaac ccaaggacac cctcatgatc tcccggaccc ccgaggtcac atgcgtggtg 1440
gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 1500
gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 1560
agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 1620
tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 1680
cgagaaccac aggtgtacac cctgccccca tcccgggacg agctgaccaa gaaccaggtc 1740
agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 1800
aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1860
ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1920
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gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc 60
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gagaaggccc caaaaagcct gatctacgcc gccagtagcc tgcagagcgg agtgcccagc 180
agattcagcg gaagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaagacttcg ccacctacta ctgccagcag tacaacatct acccctacac cttcggccag 300
ggcaccaaac tggaaatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360
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cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
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ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagttcgc ccgtcacaaa gagcttcaac aggggagagt gttga 645
<210> 3
<211> 449
<212> PRT
<213> Artificial Sequence
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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr
20 25 30
Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile
35 40 45
Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr
65 70 75 80
Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ser Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 4
<211> 214
<212> PRT
<213> Artificial Sequence
<400> 4
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
Claims (10)
1. An anti-human IL12/23 stable transgenic cell line, which is named anti-human IL12/23CHO-S stable transgenic cell line, deposited at the chinese collection of typical cultures at university of martial arts, at 3/11/2020, with the deposit number cctccc No. C202050.
2. A method for constructing an anti-human IL12/23 stable transgenic cell line, which is characterized by comprising the following steps:
(1) Preparing a plasmid containing the heavy chain gene of the anti-human IL12/23 antibody and a plasmid containing the light chain gene of the anti-human IL12/23 antibody, and co-transfecting the two plasmids into a parent CHO-S cell;
(2) Puromycin/G418 screening is carried out on the CHO-S cells after transfection to obtain stable transgenic cells;
(3) And (3) carrying out monoclonal cell strain sorting on the stable transfer cells to obtain the anti-human IL12/23 stable transfer cell strain CHO-S.
3. The method of claim 2, wherein in step (2), the Puromycin/G418 selection comprises: the transfected cells were cultured in complete medium, 48h and then the complete medium was replaced with screening medium containing 10ug/mL puromycin and 800ug/mL G418, and the screening medium was replaced every 3-4 days until the cells grew steadily.
4. The method for constructing an anti-human IL12/23 stable transgenic cell line according to claim 3, wherein the final concentration of puromycin and G418 in the screening culture solution is 10ug/mL and 800ug/mL, respectively.
5. The method for constructing an anti-human IL12/23 stable transgenic cell line according to claim 3, wherein the Puromycin/G418 screening further comprises: and inoculating the cells which grow stably in the screening culture solution into the complete culture solution for continuous culture to obtain the successfully transfected stably transfected cells.
6. The method for constructing a stable human IL12/23 transgenic cell line according to claim 5, wherein the cells stably grown in the screening medium are inoculated into the complete medium for 2 to 3 days of continuous culture, and the cell density in the complete medium after the inoculation is 5X 10 5 cells/mL。
7. The method of constructing an anti-human IL12/23 stable transgenic cell line according to claim 2, wherein in step (3), the monoclonal cell line selection comprises: and inoculating the monoclonal cell strains in the stable transfer cells into a complete culture solution one by one, continuously culturing for 12-14 days, and detecting the yield of the anti-human IL12/23 antibody of each monoclonal cell strain to obtain the anti-human IL12/23 stable transfer cell strain CHO-S.
8. Use of an anti-human IL12/23 stable transgenic cell line as described in claim 1 for the preparation of an anti-human IL12/23 antibody.
9. The use according to claim 8, comprising the steps of:
(a) Inoculating the anti-human IL12/23 stable transfer cell strain CHO-S into shake flask containing complete CD culture solution, and placing at 37deg.C, 5% CO 2 Subculturing at 120 rpm;
(b) After subculturing, transferring the cells into a large-volume culture shake flask, and inoculating cells with initial density of more than 3×10 5 cells/mL; in order to make the culture solution have higher dissolved oxygen, the volume of the culture solution in the culture shake flask is not more than 1/5 of the total volume of the culture flask;
(c) When the cell density reaches 1X 10 6 cutting off the supply of carbon dioxide after cell/mL, and regulating the rotating speed of the shaking table to 130-135 rpm;
(d) Culturing for 6-8 days, collecting culture solution, centrifuging, filtering with 0.45 μm filter membrane to obtain cell culture supernatant, and purifying and eluting cell culture supernatant sequentially to obtain anti-human IL12/23 antibody.
10. The use of an anti-human IL12/23 stable transgenic cell line as claimed in claim 1 for the preparation of a drug substance, a drug substance agent or a cosmetic substance.
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| US6914128B1 (en) * | 1999-03-25 | 2005-07-05 | Abbott Gmbh & Co. Kg | Human antibodies that bind human IL-12 and methods for producing |
| DK2168984T3 (en) * | 1999-03-25 | 2012-12-10 | Abbott Gmbh & Co Kg | Human antibodies that bind human IL-12 and methods for its production |
| ES2797126T3 (en) * | 2008-05-30 | 2020-12-01 | Xbiotech Inc | IL-1 alpha antibody uses |
| CN102167741B (en) * | 2010-02-25 | 2014-05-14 | 上海百迈博制药有限公司 | Fully human anti-TNF-alpha (Tumor Necrosis Factor-alpha) monoclonal antibody and preparation method as well as application thereof |
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