CN108998450A - Primer, cDNA, carrier, AQP4 monoclonal antibody and preparation method - Google Patents
Primer, cDNA, carrier, AQP4 monoclonal antibody and preparation method Download PDFInfo
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- CN108998450A CN108998450A CN201810893757.6A CN201810893757A CN108998450A CN 108998450 A CN108998450 A CN 108998450A CN 201810893757 A CN201810893757 A CN 201810893757A CN 108998450 A CN108998450 A CN 108998450A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention belongs to DNA recombinant technique field, a kind of primer, the cDNA of coding, carrier, AQP4 monoclonal antibody and preparation method are disclosed, the cho-cell clone of expression rat AQP4 m23 is established；With primer RT-polymerase chain reaction, the cDNA of encoding rat AQP4 m23 a kind of has been cloned from the total serum IgE extracted in rat brain；After sequencing, cDNA is inserted into the site NheI and SacII of a perres 2-egfp carrier.High-affinity of the anti-mouse AQP4 and anti-human AQP4 prepared by the present invention with AQP4 in Central nervous system in mouse, and be proved that class neuromyelitis optica lesion and symptom can be generated in body and external induction mouse.This is conducive to the research of neuromyelitis optica and new treatment is explored.
Description
Technical field
The invention belongs to DNA recombinant technique field more particularly to a kind of primer, the cDNA of coding, carrier, AQP4 monoclonals
Antibody and preparation method.
Background technique
Currently, the prior art commonly used in the trade is such thatAntigen mouse in vivo immunization: ready-made antigen or conjunction are utilized
It at polypeptide as antigen, is injected in Mice Body, the immune response by inducing mouse obtains antibody, and finally carries out antibody
Purification.Phage antibody technology: being shown expression in phage surface for all cloning of V_H gene of certain animal, benefit
Go out to carry the clone of specific antibody gene with different antigen selections, to obtain specific antibody.
In conclusion problem of the existing technology is:
(1) according to document, anti-aquaporin 4 (anti-AQP4) can not be generated in inducing mouse body by antigen immunization
Potent antibodies, that is, the antibody no pathogenicity effect generated, therefore the model foundation of neuromyelitis optica can not be applied to.
(2) antigen as required for phage antibody technology can only be obtained by way of Peptide systhesis, not had
There is the space folding structure of albumen, therefore the antibody screened can not be accomplished to carry out good combination with internal receptor, thus
It affinity and pathogenic substantially reduces.
(3) anti-aquaporin 4 antibody (anti-AQP4 antibody) at present on the market is for aquaporin 4
(AQP4) detection, however the effect for keeping animal pathogenic is not had, therefore be not used to the foundation of disease model.
Solve the difficulty and meaning of above-mentioned technical problem:
Difficulty: since by antigen immunization having for anti-aquaporin 4 (anti-AQP4) can not be generated in inducing mouse body
Imitate antibody.Therefore, to obtain the anti-AQP4 antibody that can be caused a disease in Mice Body, need to filter out to mouse AQP4 have high-affinity and
Pathogenic antibody.In the above-described techniques, since the antigen of use is complete AQP4 albumen, and gone out by immune induction
Antibody can not adapt to the space structure of the antigen completely, and antibody composition is uncontrollable, therefore the antibody prepared does not have high parent
With power and pathogenic.
And in the design, by carrying out monoclonal antibody preparation directly against rat AQP4m23, can get real in vitro
There is pathogenic anti-mouse AQP4 monoclonal antibody in testing.
Meaning: anti-AQP4 antibody is the major virulent factor of neuromyelitis optica.Neuromyelitis optica is a kind of high pathogenic
Property, high crippling, the disease of high relapse rate.The treatment method of neuromyelitis optica mature at present can not still cure and in advance completely
Anti- breaking-out.Therefore, further investigation has been both needed to for neuromyelitis optica pathogenesis and therapeutic scheme.
It is still very difficult for establishing the animal model of neuromyelitis optica at present.Anti- mouse AQP4 monoclonal antibody in the design
The rat model for helping to establish neuromyelitis optica has positive effect for the research of neuromyelitis optica.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of primer, the cDNA of coding, carrier, AQP4 Dan Ke
Grand antibody and preparation method.
The invention is realized in this way a kind of primer, the sequence of the primer is SEQ ID NO:1.
Another object of the present invention is to provide a kind of rat AQP4m23cDNA encoded using the primer.
Another object of the present invention is to provide a kind of perres2-egfp loads using the rat AQP4m23cDNA
Body, which is characterized in that the rat AQP4m23cDNA is inserted into the site NheI and SacII of perres 2-egfp carrier.
Include perres 2-egfp carrier expression rat AQP4's another object of the present invention is to provide a kind of
Chinese hamster ovary celI clone.
Another object of the present invention is to provide a kind of AQP4 monoclonal antibodies cloned by the Chinese hamster ovary celI.
Another object of the present invention is to provide a kind of preparation method of AQP4 monoclonal antibody, the AQP4 monoclonal is anti-
The preparation method of body includes: the cDNA segment quilt of mankind's AQP4m23 isomers in mouse AQP4M23 isomers or in D series
It is inserted into pruebac4.5 carrier, to produce the Pasteur baculoviral containing mAQP4 or hAQP4；It is struck in e series using AQP4
Wild-type mice is used except mouse is as host, or in d series；Mouse is intraperitoneal molten with the phosphate buffer salt containing bv
Liquid is immunized, Pasteur's baculovirus expression mAQP4-m23 or hAQP4-m23 isotope and pertussis toxin.Flow cytometry
It is used to screen the compatibility of AQP4 antibody with raji cell assay Raji；.
In conclusion advantages of the present invention and good effect are as follows:Anti- mouse AQP4 and anti-human AQP4 prepared by the present invention exist
High-affinity with AQP4 in Central nervous system in mouse, and be proved to regard in body and the external mouse generation class that induces
Neuromyelities lesion and symptom.This is conducive to the research of neuromyelitis optica and new treatment is explored.
Detailed description of the invention
Fig. 1 is the preparation method flow chart of AQP4 monoclonal antibody provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
As shown in Figure 1, the preparation method of AQP4 monoclonal antibody provided in an embodiment of the present invention the following steps are included:
S101: the cho-cell clone of expression rat AQP4m23 is established；With primer RT-polymerase chain reaction, from
The cDNA of encoding rat AQP4m23 a kind of has been cloned from the total serum IgE extracted in rat brain；
S102: after sequencing, cDNA is inserted into the site NheI and SacII of a perres 2-egfp carrier,
In unique site AflII ligatured by Linker and be rewritten into the site EcoRI；With EcoRI to the carrier before transfection
It is linearized；Chinese hamster ovary celI is converted by the carrier of linearisation, with 2 × 105Density sow on 3.5 centimetres of plate；
S103: transfecting two days later, and cell is compressed, and is re-added in 10 10 centimetres of culture dish, wherein
Contain g 418；Behind after g418 is selected about 10 days, the bacterium colony through the visible EGFP fluorescent positive of Immunofluorescence test is selected
Out；After carrying out amplification and western blotting verifying to AQP4 expression, obtained by limitation dilution several unicellular
Clone.
Mouse AQP4 of the preparation method coding of AQP4 monoclonal antibody provided in an embodiment of the present invention in E series
(mAQP4) the cDNA segment of M23 isomers or mankind AQP4 (hAQP4) m23 isomers in D series is inserted into
Pruebac4.5 carrier (life technology, carlsbad, ca, the U.S.), to produce the rod-shaped disease of the Pasteur containing mAQP4 or hAQP4
Malicious (bv).In order to avoid immune tolerance mAQP4, we use AQP4 knock-out mice as host in e series, or in d system
Wild-type mice is used in column.Mouse is immunized intraperitoneal with the phosphate buffered saline containing bv, and bv expresses mAQP4-
M23 or hAQP4-m23 isotope and pertussis toxin.Flow cytometry and raji cell assay Raji (are steadily expressed with Chinese hamster ovary celI
AQP4 (hAQP4) m1 the and m23 isomers of mAQP4-m1 and m23 and people) for screening the compatibility of AQP4 antibody；Selection
Two clones are used for NMO (neuromyelitis optica) models of mouse, because these antibody are shown to mouse m1 and m23；With
And the highest affinity of mankind m1 and mankind m23.The sequence of gene comes from webpage NCBI:https:// www.ncbi.nlm.nih.gov/gene/11829。
The present invention has cloned the cDNA of encoding rat AQP4m23 a kind of from from the total serum IgE extracted in rat brain.SEQ
ID NO:1 primer is as follows:
5 '-GCTAGCATCATGGTGGCTTTCAAAGGAGTCTGGAC-3 and
5′-CCGCGGTCATACAGAAGATAATACCTCTCCAGACG-3′。
After sequencing, cDNA is inserted into a perres 2-egfp carrier, and (clone technology in California, USA mountain scene city is real
Test room) the site NheI and SacII in, wherein unique site AflII by Linker ligation be rewritten into one EcoRI
Point.To establish the Chinese hamster ovary celI clone that can stablize expression rat AQP4, the carrier is linearized with EcoRI before transfection.
Then, Chinese hamster ovary celI is converted by the carrier of linearisation, with 2 × 105Density sow on 3.5 centimetres of plate, should during
It uses in rouge amine reagent (life technology, Carlsbad, ca, the U.S.).It is transfecting two days later, cell is compressed, and is added again
It is added in 10 10 centimetres of culture dish, wherein containing g 418 (500 mcg/mls, the nacalai tesque of kyoto, Japan
Company).Behind after g418 is selected about 10 days, the bacterium colony through the visible EGFP fluorescent positive of Immunofluorescence test is selected
Come.After carrying out amplification and westernblotting verifying to AQP4 expression, several unicellular grams have been obtained by limiting dilution
It is grand.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>First Affiliated Hospital of Kunming Medical University
<120>primer, cDNA, carrier, AQP4 monoclonal antibody and preparation method
<141> 2018-08-07
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 70
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
Claims (6)
1. a kind of primer, which is characterized in that the sequence of the primer is SEQ ID NO:1.
2. a kind of rat AQP4 m23cDNA encoded using primer described in claim 1.
3. a kind of perres 2-egfp carrier using rat AQP4 m23cDNA described in claim 2, which is characterized in that institute
Rat AQP4 m23cDNA is stated to be inserted into the site NheI and SacII of perres 2-egfp carrier.
4. a kind of Chinese hamster ovary celI clone comprising the expression of perres 2-egfp carrier described in claim 3 rat AQP4.
5. a kind of AQP4 monoclonal antibody that the Chinese hamster ovary celI as described in claim 4 is cloned.
6. a kind of preparation method of AQP4 monoclonal antibody as claimed in claim 5, which is characterized in that the AQP4 monoclonal is anti-
The preparation method of body includes: the cDNA segment quilt of mankind's AQP4m23 isomers in mouse AQP4M23 isomers or in D series
It is inserted into pruebac4.5 carrier, to produce the Pasteur baculoviral containing mAQP4 or hAQP4；It is struck in e series using AQP4
Wild-type mice is used except mouse is as host, or in d series；Mouse is intraperitoneal molten with the phosphate buffer salt containing bv
Liquid is immunized, Pasteur's baculovirus expression mAQP4-m23 or hAQP4-m23 isotope and pertussis toxin.Flow cytometry
It is used to screen the compatibility of AQP4 antibody with raji cell assay Raji.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810893757.6A CN108998450A (en) | 2018-08-08 | 2018-08-08 | Primer, cDNA, carrier, AQP4 monoclonal antibody and preparation method |
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| CN201810893757.6A CN108998450A (en) | 2018-08-08 | 2018-08-08 | Primer, cDNA, carrier, AQP4 monoclonal antibody and preparation method |
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| CN201810893757.6A Withdrawn CN108998450A (en) | 2018-08-08 | 2018-08-08 | Primer, cDNA, carrier, AQP4 monoclonal antibody and preparation method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748036A (en) * | 2020-06-28 | 2020-10-09 | 中国人民解放军总医院 | Human-mouse chimeric monoclonal antibody |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1910456A (en) * | 2003-11-25 | 2007-02-07 | 马约医学教育与研究基金会 | Marker for neuromyelitis optica |
| CN103937836A (en) * | 2014-04-04 | 2014-07-23 | 天津医科大学总医院 | Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof |
| US20160317629A1 (en) * | 2015-05-01 | 2016-11-03 | The Board Of Trustees Of The Leland Stanford Junior University | Aquaporin tolerizing vaccines and methods of use thereof |
| CN106318974A (en) * | 2016-08-29 | 2017-01-11 | 南方医科大学南方医院 | Cell immobilization technology and AQP4 antibody detection kit prepared through same |
-
2018
- 2018-08-08 CN CN201810893757.6A patent/CN108998450A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1910456A (en) * | 2003-11-25 | 2007-02-07 | 马约医学教育与研究基金会 | Marker for neuromyelitis optica |
| CN103937836A (en) * | 2014-04-04 | 2014-07-23 | 天津医科大学总医院 | Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof |
| US20160317629A1 (en) * | 2015-05-01 | 2016-11-03 | The Board Of Trustees Of The Leland Stanford Junior University | Aquaporin tolerizing vaccines and methods of use thereof |
| CN106318974A (en) * | 2016-08-29 | 2017-01-11 | 南方医科大学南方医院 | Cell immobilization technology and AQP4 antibody detection kit prepared through same |
Non-Patent Citations (5)
| Title |
|---|
| A.S.VERKMAN等: "Biology of AQP4 and anti-AQP4 antibody: therapeutic implications for NMO", 《BRAIN PATHOL》 * |
| KAORI MIYAZAKI-KOMINE等: "High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin-4 competing with the neuromyelitis optica autoantibody, NMO-IgG", 《BR J PHARMACOL》 * |
| KAZUHIRO KUROSAWA等: "Severely exacerbated neuromyelitis optica rat model with extensive astrocytopathy by high affinity anti-aquaporin-4 monoclonal antibody", 《ACTA NEUROPATHOLOGICA COMMUNICATIONS》 * |
| 俞晓燕等: "水通道蛋白4重组质粒的构建及其在体外对低糖反应的观察", 《实用医学杂志》 * |
| 杨文茜等: "水通道蛋白4的研究进展", 《中国实用医药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748036A (en) * | 2020-06-28 | 2020-10-09 | 中国人民解放军总医院 | Human-mouse chimeric monoclonal antibody |
| CN111748036B (en) * | 2020-06-28 | 2022-03-29 | 中国人民解放军总医院 | Human-mouse chimeric monoclonal antibody |
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Inventor after: Wu Yan Inventor after: Zhong Lianmei Inventor after: Geng Jia Inventor after: Wu Qian Inventor after: Lei Xiaoguang Inventor before: Wu Yan Inventor before: Zhong Lianmei Inventor before: Geng Jia Inventor before: Wu Qian Inventor before: Lei Xiaoguang |
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Application publication date: 20181214 |