CN103525823A - Preparation method of CD20 chimeric antibody for treating urticaria - Google Patents
Preparation method of CD20 chimeric antibody for treating urticaria Download PDFInfo
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- CN103525823A CN103525823A CN201310477885.XA CN201310477885A CN103525823A CN 103525823 A CN103525823 A CN 103525823A CN 201310477885 A CN201310477885 A CN 201310477885A CN 103525823 A CN103525823 A CN 103525823A
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Abstract
The invention discloses a preparation method of a CD20 chimeric antibody for treating urticaria. The preparation method comprises the following steps: A, amplifying light-chain and heavy-chain variable region genes of an anti-mouse CD20 antibody from a hybridoma cell; B, amplifying light-chain and heavy-chain constant region genes of the human antibody from the 293E cell; C, linking the light-chain and heavy-chain variable region genes of the anti-mouse CD20 antibody subjected to amplification in step A with the light-chain and heavy-chain constant region genes of the human antibody subjected to amplification in step B respectively to obtain light-chain and heavy-chain CD20 chimeric antibody genes; and D, cloning the light-chain and heavy-chain CD20 chimeric antibody genes obtained in step C to the PTY5 eukarotic expression carrier respectively. The CD20 chimeric antibody after expression has cell binding activity and is effective and safe in treating urticaria.
Description
Technical field
The present invention relates to chimeric antibody, be specifically related to a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria.
Background technology
CD20 is a kind of cytolemma cross-film phosphorprotein of non-saccharification, and it is comprised of 297 amino acid, and relative molecular mass is 35 * 10
3, be expressed in B cell surface more than 95% normal or that worsen.Be considered to B is the distinctive sign of cell surface always.The expression of CD20 antigen is strictly limited in late period and the ripe bone-marrow-derived lymphocyte of pre-B lymphocyte, in hemopoietic stem cell, progenitor cell and other healthy tissues without the expression of CD20 antigen.When bone-marrow-derived lymphocyte is divided into the plasmocyte of secretory antibody, the expression of CD20 antigen also disappears thereupon.The CD20 homology of people and mouse can reach 73%.Although it is clear that the function of CD20 is not still set forth completely, but research is found at present, CD20 antigen is a part for signal conduction mixture, and its function class is similar to calcium channel, participate in to regulate growth and the differentiation of bone-marrow-derived lymphocyte, concrete effect comprises that suppressing B cell enters the S/G2+M phase from the G1 phase; Mobilize the calcium storage vault in born of the same parents that calcium ion concn is raise; Activate tyrosine and serine/threonine protein kitase, make caspase activation.CD20 is combined rear internalization phenomenon with anti-CD20 antibodies not obvious, so cell surface CD20 molecular amounts is not because reduce in a large number with antibodies.Can there is not the phenomenon that obvious cell surface comes off in CD20, in human serum, without free CD20, exist yet.Therefore CD20 antigen is the ideal role site of B cell-targeting treatment.The B cell surface molecule CD20 of anti-CD-20 monoclonal antibody (mAb) having been take is now target, is used for the treatment of B cell lymphoma, has curative effect.
Chronic urticaria is a kind of common skin diseases, and result for the treatment of is not good at present.
Summary of the invention
Task of the present invention is to provide the preparation method after a kind of express with the CD20 chimeric antibody that is used for the treatment of urticaria of Cell binding activity, effective and safe.
The present invention realizes by following technical proposals:
A preparation method for the CD20 chimeric antibody of urticaria, is characterized in that: described in be used for the treatment of urticaria CD20 chimeric antibody comprise the following steps::
A. chain variable region gene and the heavy chain variable region gene of anti-MuCD20 antibody increase from hybridoma;
B. constant region of light chain gene and the weight chain constant area gene of people's antibody increase from 293E cell;
C. the chain variable region gene of the anti-MuCD20 antibody after the poly-A amplification of step, heavy chain variable region gene are connected with the constant region of light chain gene, the weight chain constant area gene that gather the people's antibody after B amplification through step respectively, obtain light chain CD20 chimeric antibody gene and heavy chain CD20 chimeric antibody gene;
D. will in the poly-C of step, obtain light chain CD20 chimeric antibody gene and heavy chain CD20 chimeric antibody gene is cloned into respectively PTY5 carrier for expression of eukaryon.
The further improvement project of the present invention comprises:
The concrete grammar of the poly-A of described step is: the cDNA of hybridoma of take is template, uses primer V
l-F and V
l-R the chain variable region gene that increases, uses primer V
h-F and V
h-R the heavy chain variable region gene that increases, wherein
V
lthe gene order of-F: 5'-CG
gAATTCatggattttcaggtgcagattatc-3', for
ecorI restriction enzyme site,
V
lthe gene order of-R: 5'-
cCTCCGAAtttgatttccagcttggtcccc-3', for
overlapping fragments,
V
hthe gene order of-F: 5'-CG
gAATTCatgggttggagcctcatcttgc-3', for
ecorI restriction enzyme site,
V
hthe gene order of-R: 5'-
cCTTGGCCagagacggtgaccgtcccttg-3', for
overlapping fragments;
The concrete grammar of the poly-B of described step is: the cDNA of 293E cell of take is template, uses primer C
l-F and C
l-R amplification constant region of light chain gene, uses primer C
h-F and C
h-R the weight chain constant area gene that increases, wherein
C
lthe gene order of-F: 5'-
aAATCAAAttcggaggggggaccaaggtg-3', for
overlapping fragments,
C
lthe gene order of-R: 5'-CG
gGATCCtcaacactctcccctgttgaag-3', for
bamhI restriction enzyme site,
C
hthe gene order of-F: 5'-
cCGTCTCTggccaagggaccacggtcac-3', for
overlapping fragments,
C
hthe gene order of-R: 5'-CG
gGATCCtcatttacccggagacagggag-3', for
bamhI restriction enzyme site.
In the poly-C of described step, by overlap extension pcr, realizes chain variable region gene after amplification and the connection of the constant region of light chain gene after amplification, and realize the heavy chain variable region gene after amplification and increase after the connection of weight chain constant area gene.
The concrete grammar of the poly-D of described step is: light chain gene, heavy chain gene are carried out to double digestion with restriction endonuclease EcoRI, restriction endonuclease BamHI respectively, be cloned into respectively with same enzyme and carry out in the carrier pTY5 of double digestion, be built into recombinant expression vector, the recombinant expression vector that light chain gene, heavy chain gene are built into is respectively pTY5-CD20H and pTY5-CD20L.
The cDNA of the cDNA of described hybridoma and described 293E cell obtains by following method: extract respectively the cell RNA of hybridoma and 293E cell, reverse transcription obtains cDNA ,-20 ℃ of preservations.
Described CD20 chimeric antibody, by protein A post affinity chromatography, obtains highly purified CD 20 antagonizing Chimeric antibody.
Described Hybridoma Cell Culture is in modified form quick hybridization oncocyte substratum; Described 293E cell cultures, in DMEM substratum, contains massfraction and is 10% foetal calf serum and dual anti-in DMEM substratum; Hybridoma and 293E cell are all incubated in the incubator of 37 ℃, CO in incubator
2volumetric concentration be 5%.
The present invention has the following advantages: screening is obtained on the basis of anti-CD-20 monoclonal cell strain, intend the gene fragment of its heavy chain of clone and light chain, build recombinant eukaryon expression vector, and finally express, purifying, the antibody of expressing has Cell binding activity, and treatment urticaria is safe and effective.
Accompanying drawing explanation
Fig. 1 is PCR electrophorogram,
Wherein: 1:V
l384bp; 2:C
l324bp; 3:V
h420bp; 4:C
h314bp;
5：V
L?+?C
L?708bp；6：V
H?+?C
H?734bp。
Fig. 2 is
ecorI,
bamhI double digestion electrophorogram;
M：λ-EcoT14Ⅰdigest?DNA?Marker；1：pTY5-CD20H；2：pTY5-CD20L。
Fig. 3 is PCR evaluation figure;
1：V
L?+?C
L?708bp；2：V
H?+?C
H?734bp。
Fig. 4 is that SDS-PAGE detects antibody figure.
Fig. 5 is the Flow cytometry figure that expresses antibody.
Embodiment
Below in conjunction with accompanying drawing, a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria of the present invention is further described:
Embodiment 1:
Primer light for mouse, weight chain variabl area sequence is synthesized in A, design of primers design; Design is synthetic for primer light, CH.
V
lthe gene order of-F: 5'-CG
gAATTCatggattttcaggtgcagattatc-3', for
ecorI restriction enzyme site,
V
lthe gene order of-R: 5'-
cCTCCGAAtttgatttccagcttggtcccc-3', for
overlapping fragments,
V
hthe gene order of-F: 5'-CG
gAATTCatgggttggagcctcatcttgc-3', for
ecorI restriction enzyme site,
V
hthe gene order of-R: 5'-
cCTTGGCCagagacggtgaccgtcccttg-3', for
overlapping fragments;
C
lthe gene order of-F: 5'-
aAATCAAAttcggaggggggaccaaggtg-3', for
overlapping fragments,
C
lthe gene order of-R: 5'-CG
gGATCCtcaacactctcccctgttgaag-3', for
bamhI restriction enzyme site,
C
hthe gene order of-F: 5'-
cCGTCTCTggccaagggaccacggtcac-3', for
overlapping fragments,
C
hthe gene order of-R: 5'-CG
gGATCCtcatttacccggagacagggag-3', for
bamhI restriction enzyme site.
V wherein
l--chain variable region gene; C
l--constant region of light chain gene; V
h-heavy chain variable region gene; C
h-weight chain constant area gene.
B, cell cultures Hybridoma Cell Culture are in modified form quick hybridization oncocyte substratum; 293E cell cultures is in DMEM substratum, and NS-1 cell cultures, in RPMI 1640 substratum, all contains massfraction and be 10% foetal calf serum and dual anti-; Be incubated in 37 ℃ of incubators, it is 5% CO that incubator contains volume fraction
2.
C, RNA extract synthetic hybridoma and the 293E cell collected respectively with cDNA, specification sheets by TRIZOL extracts cell total rna, survey OD and electrophoresis detection RNA quality, by RT-PCR test kit specification sheets, carry out reverse transcription and obtain cDNA, be stored in-20 ℃ standby.
D, antibody are light, heavy chain gene transfer with the cDNA that clones to derive from hybridoma to be template, to use primer V
l-F and V
l-R, V
h-F and V
h-R increases respectively gently, heavy chain variable region gene; The cDNA that derives from 293E cell of take is template, uses primer C
l-F and C
l-R, C
h-F and C
h-R increases respectively gently, weight chain constant area gene.After above-mentioned sequence sequence verification is correct, by overlap extension pcr, connect variable region and constant region, obtain complete antibody gene, as shown in Figure 1, amplification size is correct for result, and after sequence verification, sequence is shown as antibody gene.Sequence verification, then incites somebody to action light, heavy chain gene restriction endonuclease again
ecorI,
bamhI carries out respectively double digestion, is cloned into respectively with same enzyme and carries out in the carrier pTY5 of double digestion, and result as shown in Figure 2, is cloned into after expression vector, and double digestion is identified errorless.Sequencing result demonstration, institute's cloned sequence is that mouse variable region of light chain adds people's constant region of light chain, the antibody constant region light chain of transferring is κ type; Another fragment is that mouse variable region of heavy chain adds people's CH, and the antibody constant region heavy chain of transferring is IgG1 type.Connect product and transform TOP10 competent cell, after dull and stereotyped (ammonia benzyl resistance) 37 ℃ of incubated overnight, picking mono-clonal identification of strains order-checking respectively.The recombinant expression vector building is called after pTY5-CD20H and pTY5-CD20L respectively.
E, transfection and evaluation transfection inoculating cell the day before yesterday: with after trysinization 293E cell, adjusting cell concn is 2.5 * 10
5individual/ml, every hole 1.0 ml inoculate 24 orifice plates, put 37 ℃ containing 5% CO
2incubator is cultured to cell attachment growth and reaches floorage 70%.Transfection is undertaken by Lipofectamine 2000 specification sheetss, and plasmid transfection amount is 1 μ g, cultivates after 4 hours and changes DMEM nutrient solution, continues to cultivate 48 hours, and then collecting cell extracts RNA, carries out reverse transcription, and PCR evaluation antibody gene is transcribed situation.As shown in Figure 3, showing can specific amplified object fragment for PCR result.This explanation carries that antibody is light, the expression vector of heavy chain gene success transfection is entered 293E cell, and can be expressed antibody gene.
F, protein expression and purification and rough determination plasmid transfection 293E cell, shake-flask culture, 4-7 days results substratum supernatants, ProteinA column purification antibody.Working method is carried out with reference to specification sheets, after purifying, by spectrophotometric instrumentation OD value, calculate concentration, with SDA-PAGE, measure purity, the molecular weight of albumen, SDS-PAGE result under reductive condition as shown in Figure 4, show 2 bands, molecular weight is about 23kDa and 51kDa, is consistent with the weight chain molecular weight of design, shows successfully to have obtained protein
Complete antibody molecule for IgG1 type.
The ELISA that G, chimeric antibody are expressed detects and resists in 4 ℃ of coated elisa plates more and spend the night with goat-anti people κ, with the PBST containing 1% BSA, at 37 ℃, seals 2 hours.Add respectively the supernatant of cleer and peaceful empty plasmid transfection in the expression of chimeric antibody, using human IgG reference material as positive control.With PBST, wash 5 times after hatching 30min for 37 ℃, then add the goat-anti people Fc antibody of HRP mark, with PBST, wash 5 times after hatching 30min for 37 ℃.Add TMB colour developing, microplate reader reads 450/630 dual wavelength reading again.After transfection 3 days, sandwich ELISA is measured the content of chimeric antibody in supernatant, by with positive reference material comparison, in supernatant, the content of antibody is between 15-24 ng/ml.
H, Flow cytometry antibody binding activity are collected NS-1 cell, every pipe 5 * 10
5individual, with PBS, wash one time centrifugal collecting cell; Each pipe adds expression antibody, and room temperature reaction 1 hour, arranges negative control; Centrifugal collecting cell, washes one time with PBS, adds two anti-FITC-goat anti-mouse iggs (1:500), room temperature lucifuge reaction 1 hour; Centrifugal collecting cell, washes one time with PBS, and each effective 300 μ l PBS are resuspended, upper machine testing; Flow cytometer detection result as shown in Figure 5, result show cotransfection mosaic gene cells and supernatant can with NS-1 Cell binding, there is parent mouse source antibodies effect.This shows that expressed embedding platform antibody has correct antigenic binding property, well and contain people's antibody constant region sequence.
The present embodiment has successfully built the chimeric antibody gene containing He Ren source, variable region, mouse source constant region, in 293E cell, carry out after transient expression, RT-PCR shows that antibody gene has carried out successfully transcribing in cell, show that CD20 chimeric antibody has Cell binding activity, treatment urticaria is safe and effective, ELISA measures antibody expression amount between 15-24 ng/ml, the CD 20 antagonizing Chimeric antibody of flow cytometer detection result confirmation cell conditioned medium secretion can be combined with NS-1 cell strain, and obtain highly purified CD 20 antagonizing Chimeric antibody by protein A post affinity chromatography, the mechanism of action that can be used for further Effect of Anti CD20 mab treatment chronic urticaria.
Claims (7)
1. a preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria, is characterized in that comprising the following steps:
Chain variable region gene and the heavy chain variable region gene of anti-MuCD20 antibody increase from hybridoma;
Constant region of light chain gene and the weight chain constant area gene of people's antibody increase from 293E cell;
The chain variable region gene of the anti-MuCD20 antibody after the poly-A amplification of step, heavy chain variable region gene are connected with the constant region of light chain gene, the weight chain constant area gene that gather the people's antibody after B amplification through step respectively, obtain light chain CD20 chimeric antibody gene and heavy chain CD20 chimeric antibody gene;
To in the poly-C of step, obtain light chain CD20 chimeric antibody gene and heavy chain CD20 chimeric antibody gene is cloned into respectively PTY5 carrier for expression of eukaryon.
2. a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria according to claim 1, is characterized in that: the concrete grammar of the poly-A of described step is: the cDNA of hybridoma of take is template, uses primer V
l-F and V
l-R the chain variable region gene that increases, uses primer V
h-F and V
h-R the heavy chain variable region gene that increases, wherein
V
lthe gene order of-F: 5'-CG
gAATTCatggattttcaggtgcagattatc-3', for
ecorI restriction enzyme site,
V
lthe gene order of-R: 5'-
cCTCCGAAtttgatttccagcttggtcccc-3', for
overlapping fragments,
V
hthe gene order of-F: 5'-CG
gAATTCatgggttggagcctcatcttgc-3', for
ecorI restriction enzyme site,
V
hthe gene order of-R: 5'-
cCTTGGCCagagacggtgaccgtcccttg-3', for
overlapping fragments;
The concrete grammar of the poly-B of described step is: the cDNA of 293E cell of take is template, uses primer C
l-F and C
l-R amplification constant region of light chain gene, uses primer C
h-F and C
h-R the weight chain constant area gene that increases, wherein
C
lthe gene order of-F: 5'-
aAATCAAAttcggaggggggaccaaggtg-3', for
overlapping fragments,
C
lthe gene order of-R: 5'-CG
gGATCCtcaacactctcccctgttgaag-3', for
bamhI restriction enzyme site,
C
hthe gene order of-F: 5'-
cCGTCTCTggccaagggaccacggtcac-3', for
overlapping fragments,
C
hthe gene order of-R: 5'-CG
gGATCCtcatttacccggagacagggag-3', for
bamhI restriction enzyme site.
3. a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria according to claim 2, it is characterized in that: in the poly-C of described step, by overlap extension pcr, realizes the chain variable region gene after amplification and increase after the connection of constant region of light chain gene, and the connection of the heavy chain variable region gene after realization amplification and the weight chain constant area gene after amplification, obtain CD20 chimeric antibody.
4. according to a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria described in claim 2 or 3, it is characterized in that: the concrete grammar of the poly-D of described step is: light chain gene, heavy chain gene are carried out to double digestion with restriction endonuclease EcoRI, restriction endonuclease BamHI respectively, be cloned into respectively with same enzyme and carry out in the carrier pTY5 of double digestion, be built into recombinant expression vector, the recombinant expression vector that light chain gene, heavy chain gene are built into is respectively pTY5-CD20H and pTY5-CD20L.
5. a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria according to claim 4, it is characterized in that: the cDNA of the cDNA of described hybridoma and described 293E cell obtains by following method: the cell RNA that extracts respectively hybridoma and 293E cell, reverse transcription obtains cDNA ,-20 ℃ of preservations.
6. a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria according to claim 5, is characterized in that: described CD20 chimeric antibody, by protein A post affinity chromatography, obtains highly purified CD 20 antagonizing Chimeric antibody.
7. a kind of preparation method who is used for the treatment of the CD20 chimeric antibody of urticaria according to claim 5, is characterized in that: described Hybridoma Cell Culture is in modified form quick hybridization oncocyte substratum; Described 293E cell cultures, in DMEM substratum, contains massfraction and is 10% foetal calf serum and dual anti-in DMEM substratum; Hybridoma and 293E cell are all incubated in the incubator of 37 ℃, CO in incubator
2volumetric concentration be 5%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000027428A1 (en) * | 1998-11-09 | 2000-05-18 | Idec Pharmaceuticals Corporation | Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-cd20 antibody |
| CN1718587A (en) * | 2005-07-11 | 2006-01-11 | 中国人民解放军军事医学科学院生物工程研究所 | A kind of CD 20 antagonizing Chimeric antibody |
-
2013
- 2013-10-14 CN CN201310477885.XA patent/CN103525823A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000027428A1 (en) * | 1998-11-09 | 2000-05-18 | Idec Pharmaceuticals Corporation | Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-cd20 antibody |
| CN1718587A (en) * | 2005-07-11 | 2006-01-11 | 中国人民解放军军事医学科学院生物工程研究所 | A kind of CD 20 antagonizing Chimeric antibody |
Non-Patent Citations (2)
| Title |
|---|
| 梁碧华 等: "CD20嵌合抗体的构建、表达及纯化的实验研究", 《中国医药导报》, vol. 9, no. 32, 30 November 2012 (2012-11-30) * |
| 梁碧华 等: "抗CD20单抗的研究进展及其临床应用", 《国际皮肤性病学杂志》, vol. 37, no. 2, 31 March 2011 (2011-03-31), pages 80 - 83 * |
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Application publication date: 20140122 |