CN102559604A - Monoclonal antibody having antagonistic action on HMGB1 B box and application thereof - Google Patents
Monoclonal antibody having antagonistic action on HMGB1 B box and application thereof Download PDFInfo
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Abstract
The invention relates to a hybridoma cell line capable of continuously and stably secreting a monoclonal antibody having antagonistic action on HMGB1 B box, and the secreted antibody. As an important inflammation medium, HMGB1 has proinflammatory and chemotactic functions, and widely takes part in pathogenesis of a plurality of inflammatory diseases, and B box is the biological activity area of HMGB1. The monoclonal antibody prepared with the method has the characteristics as follows: the monoclonal antibody is IgG2a, kappa, and can be specifically combined with vertebrate HMGB1 B box to inhibit the biological activity; furthermore, the monoclonal antibody can be used for western-blot and ELISA tests. In conclusion, the monoclonal antibody can be used for detecting vertebrate HMGB1 B box on one hand, and also can be used for therapy on the other hand.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of monoclonal antibody and range of application thereof of this important inflammatory mediator of antagonism HMGB1 B box.
Background technology
(high mobility group protein B1 HMGB1) is one of high mobility group protein (HMG) family member to high mobility group protein B 1, gains the name because of in polyacrylamide gel electrophoresis (SDS-PAGE), having high transfer ability.HMGB1 is distributed widely in the tissues such as Lymphoid tissue, brain, liver, lung, the heart, spleen, kidney, and HMGB1 is present in karyon except that in liver, cerebral tissue, mainly being present in the endochylema in the great majority tissue.HMGB1 is its aminoacid sequence high conservative during evolution, and rodent and people's amino acid sequence homology is up to more than 98%, and mouse and rat amino acid sequence homology are especially up to 100%.Human HMGB1 gene is positioned on the 13q12 karyomit(e), comprises 5 exons and 4 introns, and coding contains 215 amino acid whose albumen, is made up of three zones: two positive charge zones (A box and B box) and a negative charge C-terminal (acidic ending).A box (aa1～79) and B box (aa89～163) are the non-specific binding districts of DNA.Wherein preceding 20 amino acid of B box are critical sites of its performance cytokine activity, can induce scavenger cell to secrete more proinflammatory cytokine such as TNF-α, or BMDC secretion IL-6.HMGB1 has in born of the same parents that regulatory gene is transcribed, the function of firm karyon structure.HMGB1 can be through activatory scavenger cell, BMDC active secretion or necrosis in the born of the same parents, apoptotic cells is passive discharges into outside the born of the same parents.Be discharged into the outer HMGB1 of born of the same parents through turning into end product acceptor (receptor for advanced glycation end products with its part height glycan; RAGE) and Toll appearance acceptor (Toll like receptor; TLR) combine; Promote the expression of the proinflammatory factor, chemokine and mhc class ii molecule on the one hand, and then participate in CD4
+The regulation and control of T cells whose development and immunne response; One side HMGB1 is as important inflammatory mediator in late period in addition; Participate in causing a disease and tissue injury and reparation of diseases such as septicemia, cardiovascular system diseases, tumour, autoimmune disorder, ischemia/reperfusion injury; In with HMGB1 can be effectively the symptom of the above disease of alleviation; Therefore, HMGB1 becomes inflammatory disease potential treatment target position.And the monoclonal antibody of antagonism HMGB1 B box can be effectively in and HMGB1, suppress its performance biological effect.
Summary of the invention
HMGB1 is causing a disease of important many inflammatory diseases of inflammatory mediator wide participation; Its BA site is positioned at B box zone; In with HMGB1 amelioration of inflammation disease effectively, the monoclonal antibody that therefore prepares antagonism HMGB1 B box is significant for the treatment of diseases of the biological effect of research HMGB1 and inflammation.
The invention provides the anti-HMGB1 B box cell strain of monoclonal antibody 2D4E3A2 of secretion, its preserving number is CGMCC No.5534.
The anti-HMGB1 B of secretion of the present invention box cell strain of monoclonal antibody 2D4E3A2 by the B box protein immunization BALB/c mouse of prokaryotic expression after separating Morr. cell and murine myeloma cell SP2/0 merge and make.
Description of drawings
Below in conjunction with accompanying drawing the present invention is described further.
Fig. 1 is the proteic expression and purification of used immunogen HMGB1 B box among the present invention.M: be albumen marker; Blank: as to be empty carrier; 1: the full bacterium liquid of transformed bacteria; 2: wash effusive liquid; 3: wash last effusive liquid; 4-6: be the HMGB1B box albumen behind the purifying.
Fig. 2 is the immunologic process synoptic diagram.
Fig. 3 is a 2D4E3A2 clone excretory monoclonal antibody hypotype synoptic diagram.1, this monoclonal antibody hypotype; 2, hypotype is identified map; 3, this monoclonal antibody light chain type; 4, light chain type identification map.
Fig. 4 is that the monoclonal antibody of 2D4E3A2 emiocytosis is used for ELISA detection synoptic diagram.
Fig. 5 is that LPS inducing mouse pyemia model detects blood plasma HMGB1 at the ELISA that 4h, 12h, 24h heart take a blood sample, utilize the monoclonal antibody of 2D4E3A2 emiocytosis to set up respectively.*p<0.01 。
Fig. 6 utilizes the monoclonal antibody of 2D4E3A2 emiocytosis to adopt Western blot method to detect scavenger cell HMGB1 expression level.
The antibody of Fig. 7 2D4E3A2 emiocytosis can be effectively alleviation myocarditis induced myocardial injury, on behalf of control group E, C represent experimental group E-B to represent 2D4E3A2 antibody intervention group.
Cell strain 2D4E3A2 of the present invention is submitted to China Committee for Culture Collection of Microorganisms common micro-organisms center on November 30th, 2011, the classification called after is secreted anti-HMGB1 B box cell strain of monoclonal antibody, and deposit number is CGMCC No.5534.The depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Embodiment
Immunogenic preparationThe albumen of HMGB1 B box adopts and is produced by prokaryotic expression based on engineering, and HMGB1 B box gene order is distinguished as follows with corresponding protein sequence:
HMGB1 B box gene order is shown in SEQ ID NO:1.
With the corresponding HMGB1 B of gene box protein sequence shown in SEQ ID NO:2.
<b >Animal immune</b>(the preparation process is: will be connected conversion with expression vector pET-28 (doctor's Shi Xiaoju present) from the fragment that pQE-80L (available from Third Military Medical University) downcuts B box gene with the B box albumen of 10 μ g purifying<i >E. coli</i>DH5 α competent cell.The single conversion bacterium colony of picking is inoculated in the LB substratum that contains kantlex, extracts DNA in a small amount with alkaline lysis.Identify with Kpn I, Hind III (the precious biotechnology ltd of giving birth in Dalian) double digestion and PCR.Get positive transformed bacteria and be inoculated in the LB substratum that contains kantlex, in 37 ℃ of jolting overnight cultures.Then, get this bacterium liquid 100 μ l and be inoculated in (sky, Hangzhou and microorganism reagent ltd) in the 6 ml LB substratum, in 37 ℃ of shaking culture to 0.5 < OD600 < 0.6 (about 2～3 h).Add isopropyl-(isopropy-β-D-thiogalactoside; IPTG) (fine biological ltd of Suzhou section) to final concentration is 1 mmolL; Behind 37 ℃ of shaking culture 6h; Collect that bacterium liquid is centrifugal to carry out SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electropheresis SDS-PAGE) analyzes (separation gel of PAGE is 150g/L), and SDS-PAGE result sees Fig. 1; At about 10000 places of Mr 1 dense especially protein band is arranged, its size is suitable with expected result.Induce pET-28 empty carrier transformed bacteria then not see this band with IPTG.Confirmation pET-28-HMGB1 B box fusion rotein is expressed in transformed bacteria successfully, sees the band (M: be albumen marker of 4-6 position among Fig. 1; Blank: as to be empty carrier; 1: the full bacterium liquid of transformed bacteria; 2: wash effusive liquid; 3: wash last effusive liquid; 4-6: be the HMGB1B box albumen behind the purifying); Protein purification post with the His mark carries out purifying, mixes with the Fu Shi Freund's complete adjuvant behind the purifying and fully after the emulsification, through subcutaneous multi-point injection immunity BALB/c mouse.Carry out the 2nd immunity at a distance from 2-3 Zhou Tongfa, after the week, through tail vein booster immunization (detailed step is seen Fig. 2).
Cytogamy and hybridoma screeningThe fusion of cell is undertaken by the method for routine, and the screening of positive cell strain adopts indirect ELISA to carry out.(detailed step is following :)
1. the preparation of feeder cell:
The preparation of Turnover of Mouse Peritoneal Macrophages: get 6～10 all BALB/c mouses in age (purchase of Yangzhou Experimental Animal Center), short neck is put to death, and is soaked in 75% alcohol, sterilizes 3～5 minutes; Cut off skin with sterile scissors, expose peritonaeum; Inject 2ml left and right sides NS with asepsis injector, flushing repeatedly, sucking-off washing fluid; Put into the 10ml centrifuge tube, centrifugal 5～6 minutes of 500-800r/min abandons most supernatant; The HAT nutrient solution that contains 20% calf serum with 50ml is processed suspension; Add 24 orifice plates, and 500 μ l/ holes (4 blocks of plates, 50ml); Put into 37 ℃ of CO
2Incubator is cultivated.
2. immune spleen cell
The preparation of splenocyte suspension: get mice immunized, pluck eyeball blood sampling, and the positive control of separation of serum during as antibody test; Disconnected simultaneously neck is put to death mouse, is soaked in 75% ethanol 5 minutes, in aseptic plate; Take out spleen; The full nutrient solution that toos many or too much for use is washed once, in the horizontalization ware on the nylon screen, counts after grinding to form cell suspension with the syringe nook closing member.
Myeloma cell's preparation: under aseptic condition, take out tumour, peel outside color part off, leave and take white portion, the full nutrient solution that toos many or too much for use is washed once, in the horizontalization ware on the nylon screen, counts after grinding to form cell suspension with the syringe nook closing member.
3. cytogamy
(1) (general spleens cell number was less in the 50ml plastic centrifuge tube by 1: 9 mixed with the above-mentioned myeloma cell who has counted and splenocyte; Can exhaust and put the 50ml centrifuge tube; As long as add the splenocyte pipes to the oncocytes of all splenocyte amounts with 9 times) the full nutrient solution that toos many or too much for use is supplemented to 45ml; 1000rpm, 8 minutes.
(2) abandon supernatant, (directly use up supernatant rapidly, the pipe end is got final product) up.
(3) at the bottom of the attack centrifuge tube, the abundant mixing of cell precipitation is disperseed.
(4) in transferring to 37 ℃ water in advance, merge: promptly in the 1000ml beaker, add the hot water of 40 ℃ of 500-600ml, and before fusion, put in 37 ℃ of water baths temperature in advance (put with TM in the water of beaker and measure temperature) earlier.During fusion:
The 50ml centrifuge tube that 1. 1:9 splenocyte and oncocyte mixed solution will be housed is put into the beaker that 37 ℃ of water of 500ml are housed, and shakes the limit in water surface top and adds 1ml 50%PEG, adds (dropwise add when adding PEG, from slow to fast, added in 1 minute) in 1 minute.
2. still in water-bath, continue to shake, wait 1 minute (promptly continuing to shake 1 minute).
3. continue to shake and limit edged adding 1ml serum-free RPMI1640 substratum, added in 1 minute.Continue distant, wait 1 minute after, add 5ml serum-free RPMI1640 substratum (the limit edged shakes, and adds in 3 minutes).
4. replenish serum-free RPMI1640 substratum to the 50ml scale, centrifugal 8 minutes of 800r/min.
5. abandon supernatant (the same) with abandoning most liquid at the bottom of the test tube up; Beat even sedimentation cell gently; Add HAT substratum 96-100ml (promptly in the centrifuge tube behind the adding 50mlHAT substratum; Cell suspension in the centrifuge tube is moved to mixing in the 100ml bottle that remains 45-50ml HAT substratum, divide and plant, every hole 1ml) in 24 well culture plates
6. 37 ℃, 5%CO
2Incubator is cultivated.
4. secrete the cell hole of the antibody that anti-HMGB1 is arranged in the screening indirect elisa method of the positive cell clone screening cells and supernatant.
The cloning of hybridoma with the positive cell hole carry out colony screening go out can the anti-HMGB1 B of stably excreting box monoclonal antibody cell strain, called after 2D4E3A2.
The monoclonal antibody of emiocytosis is tired, the evaluation of atopic and hypotypeAdopt indirect elisa method to measure the tiring of mAb, Western blot method and carry out that specificity is identified, monoclonal antibody subtype identifying test paper bar (the The IsoQuick strips of sigma aldrich; ISOQ25) carry out the evaluation of monoclonal antibody hypotype; Its operation steps is carried out according to specification sheets, and the result sees Fig. 3.Can find out that by Fig. 3 the secreted monoclonal antibody of 2D4E3A2 cell is IgG2a, the κ type, (1, the hypotype of monoclonal antibody to be checked; 2, hypotype is identified map; 3, this monoclonal antibody light chain type; 4, light chain type identification map).The stable back of going down to posterity of hybridoma cell strain is frozen in liquid nitrogen container, still can secrete mAb effectively after the recovery.
The application of the monoclonal antibody of emiocytosisThe monoclonal antibody of 2D4E3A2 emiocytosis is used for the detection (restriction of no kind) of vertebrates HMGB1 through ELISA (double-antibody sandwich), Western blot; In addition, the monoclonal antibody of 2D4E3A2 emiocytosis can be used for myocarditic treatment.
Application example is following:
1. the monoclonal antibody of 2D4E3A2 emiocytosis is used for the detection of mice plasma HMGB1
Mouse peritoneal injection LPS (10mg/kg) is respectively at 4h, 12h, the heart blood sampling of 24h anesthesia back, separated plasma; Join in 96 orifice plates that encapsulate this antibody in advance; 4 ℃ of overnight cultures, the washing back adds the polyclonal antibody (available from Abcam company) of the anti-HMGB1 of rabbit, and the washing back adds the HRP-IgG antibody of goat antirabbit; The washing back adds TMB, surveys its OD value (method of use of this monoclonal antibody is seen Fig. 4).Like Fig. 5, behind the mouse peritoneal injection LPS24h, the HMGB1 level reaches 200 ± 67.3ng/ml in the serum, compares obvious increase with control group.
2. the monoclonal antibody of 2D4E3A2 emiocytosis utilizes Western blot method to detect the expression of scavenger cell HMGB1
Separate Turnover of Mouse Peritoneal Macrophages and plant in 24 orifice plates, LPS stimulated 1 hour, changed nutrient solution after the washing; Continue culturing cell, collecting cell after 24 hours, boils at cracking; Carry out the SDS-PAGE electrophoresis, carry out immunoblot experiment behind the commentaries on classics film, the WB method detects the expression of scavenger cell HMGB1.Have Fig. 6 to find out, the monoclonal antibody of preparation (1D2F3E4,2D4E3A2) with available from having a special reaction in the antibody of Abcam is the same.
3. the monoclonal antibody of 2D4E3A2 emiocytosis is used for myocarditic treatment
100 μ g mouse cardiac muscle sphaeroprotein (MyHC-α
614-629, Ac-SLKLMATLFSTYASAD-OH) mix with Fu Shi Freund's complete adjuvant (CFA) 1:1, respectively at the 0th and 7 day, immune mouse, control group carries out immunity with CFA and PBS; Be divided into two groups a week after the immunity for the second time.PBS is used in one group of HMGB1 B box monoclonal antibody subcutaneous injection with 100 μ g/mouse, other one group, 2 week the back put to death mouse, collect serum, formalin fixed heart tissues; Hematoxylin-eosin (H-E) dyeing.The infiltration degree of myocardial damage and inflammatory cell, by two pathologist adopt double-blind methods according to the Dallas Case definition classification carry out.Case definition is following:
" 0 ", do not have an inflammatory infiltration;
" 1 ", a small amount of inflammatory cell infiltration of cardiac muscular tissue;
" 2 ", focal inflammation cellular infiltration, inflammatory cell infiltration quantity is greater than 100;
" 3 ", the infiltration of myocardium cell necrosis and inflammatory cell appears greater than 10% myocardium section;
" 4 ", the infiltration of myocardium cell necrosis and inflammatory cell appears greater than 30% myocardium section.
Inject the monoclonal antibody group like Fig. 7, the myocardial damage degree obviously alleviates.
SEQUENCE LISTING
< 110>Jiangsu University
< 120>monoclonal antibody and the application thereof of antagonism HMGB1 B box
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 225
<212> DNA
< 213>artificial sequence
<400> 1
ttcaaggatc ccaatgcacc caagaggcct ccttcggcct tcttcctctt ctgctctgag 60
tatcgcccaa aaatcaaagg agaacatcct ggcctgtcca ttggtgatgt tgcgaagaaa 120
ctgggagaga tgtggaataa cactgctgca gatgacaagc agccttatga aaagaaggct 180
gcgaagctga aggaaaaata cgaaaaggat attgctgcat atcga 225
<210> 2
<211> 75
<212> PRT
< 213>artificial sequence
<400> 2
Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr
35 40 45
Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg
65 70 75
Claims (2)
1. secrete anti-HMGB1 B box cell strain of monoclonal antibody 2D4E3A2, its preserving number is CGMCC No.5534.
The anti-HMGB1 B of the described secretion of claim 1 box cell strain of monoclonal antibody 2D4E3A2 by the B box protein immunization BALB/c mouse of prokaryotic expression after separating Morr. cell and murine myeloma cell SP2/0 merge and make.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114957440A (en) * | 2022-05-26 | 2022-08-30 | 江苏大学 | Recombinant HMGB 1A Box protein and obtaining method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101132811A (en) * | 2004-10-22 | 2008-02-27 | 米迪缪尼股份有限公司 | High affinity antibodies against HMGB1 and methods of use thereof |
| CN101628942A (en) * | 2009-07-24 | 2010-01-20 | 中国人民解放军第三军医大学 | Recombinant fusion protein based on human TMPD1 and HMGB1 A box and preparation method thereof |
| CN101691580A (en) * | 2009-09-28 | 2010-04-07 | 中国人民解放军第三军医大学 | Novel fusion protein for human HMGB1 A box and acidic tail and use thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101132811A (en) * | 2004-10-22 | 2008-02-27 | 米迪缪尼股份有限公司 | High affinity antibodies against HMGB1 and methods of use thereof |
| CN101628942A (en) * | 2009-07-24 | 2010-01-20 | 中国人民解放军第三军医大学 | Recombinant fusion protein based on human TMPD1 and HMGB1 A box and preparation method thereof |
| CN101691580A (en) * | 2009-09-28 | 2010-04-07 | 中国人民解放军第三军医大学 | Novel fusion protein for human HMGB1 A box and acidic tail and use thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114957440A (en) * | 2022-05-26 | 2022-08-30 | 江苏大学 | Recombinant HMGB 1A Box protein and obtaining method and application thereof |
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Inventor after: Su Zhaoliang Inventor after: Wang Shengjun Inventor after: Xu Huaxi Inventor after: Zhou Chenglin Inventor after: Yang Fengying Inventor after: Li Ping Inventor after: Sun Caixia Inventor after: Sun Fanzhi Inventor after: Liu Yanfang Inventor after: Zheng Dong Inventor after: Tong Jia Inventor before: Su Zhaoliang Inventor before: Zhou Chenglin Inventor before: Sun Caixia Inventor before: Sun Fanzhi Inventor before: Liu Yanfang Inventor before: Zheng Dong Inventor before: Tong Jia Inventor before: Wang Shengjun Inventor before: Xu Huaxi |
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Free format text: CORRECT: INVENTOR; FROM: SU ZHAOLIANG ZHOU CHENGLIN SUN CAIXIA SUN FANZHI LIU YANFANG ZHENG DONG TONG JIA WANG SHENGJUN XU HUAXI TO: SU ZHAOLIANG ZHOU CHENGLIN YANG FENGYING LI PING SUN CAIXIA SUN FANZHI LIU YANFANG ZHENG DONG TONG JIA WANG SHENGJUN XU HUAXI |
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