CN101891817B - Monoclonal antibody capable of preventing high mobility group box-1 (HMGB-1) and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody capable of preventing high mobility group box-1 (HMGB1) and an application thereof. The heavy chain variable region of the monoclonal antibody is provided with an amino acid residue basic sequence as shown in sequence 1 in a sequence table, and the light chain variable region thereof is provided with an amino acid residue basic sequence as shown in sequence 2 in the sequence table. Experiments prove that the dissociation constants of the monoclonal antibody secreted by a hybridoma cell strain 3B-1 CGMCC No.2907, a single chain antibody 3B-1scFV derived from the monoclonal antibody, a human-rat chimeric antibody ch-3B1 or a Fab fragment and a human HMGB1 are respectively 7.8nM, 100nM, 7.8nM and 40nM. The monoclonal antibody and derivatives thereof can recognize the HMGB1 in a mode of high affinity and specificity, neutralize the biological activity of the HMGB1, can be used for curing ichorrhemia, systemic inflammatory response syndrome or acute lung injury and other inflammatory reactions, can be used for curing rheumatoid arthritis, diabetes mellitus or lupus erythematosus and other autoimmune diseases and also can be used for clinically inspecting the level of the HMGB1.

Description

The monoclonal antibody of resisting high mobility group protein B-1 and application thereof

Technical field

The present invention relates to monoclonal antibody and the application thereof of resisting high mobility group protein B-1.

Background technology

(high mobility group box I) is one of the member of high mobility family in the chromosomin to HMGB-1.HMGB-1 be one by 219 amino-acid residues constitute, molecular weight is the single chain polypeptide of 30kDa, its express general and also amount also bigger.Albumen can be divided into three parts: two homologous DNA calmodulin binding domain CaMs (Boxes A and B) and acid C-terminal.HMG boxes A is the L type structure that is formed by 80 similar amino acid fragments with B.

HMGB-1 is incorporated in the ditch of double-stranded DNA, combining site and unrelated dna sequence, but closely related with its structure, be usually located at nodular DNA, curved DNA or right-angled intersection point place.HMGB-1 is curved DNA promptly, facilitates the formation of nucleoprotein complex body, so facilitate DNA conjugated protein with its interaction in corresponding site separately.Can not directly combine with straight line DNA like the p53 transcription factor, only after HMGB-1 combined with DNA earlier and makes its bending, p53 could combine in corresponding with it effectively site, and HMGB-1 spins off from complex body then.HMGB1-/-mouse defectiveness in the activation of GR response gene, therefore can after birth, just die soon because of hypoglycemia, this result has further supported the conclusion of HMGB-1 as transcriptional regulator.

HMGB-1 is present in the intercellular substance of neurocyte, the growth of the projection that can excite nerve.HMGB1 stimulates the migration of smooth muscle cell (SMC) and protofibril cell, can also cause that the reorganization of actin cytoskeleton and the form of sports type cell change.In addition, HMGB-1 is relevant with the plasminogen activation system, therefore in the regulation and control of extracellular protein hydrolysis, cell migration, inflammation, fibrinolysis, wound healing and tumour invasion, plays a role.

HMGB-1 does not have signal sequence, and its secretion process is not through classical " rough surfaced endoplasmic reticulum-golgi body " approach.In the activatory monocyte, HMGB-1 is released through cell exocrine through a redistribution process to secretory vesicle in examine then.

Research shows, stimulates the RAW264.7 cell with LPS, and composition changes in Using SDS-PAGE analysis of cells nutrient solution, and after 18 hours, finding to have molecular weight is the albumen appearance of 30-KD.N terminal sequence according to this factor is analyzed, and it is belonged to HMG-1, and a kind of DNA bonded is nonhistones.Be renamed as HMGB-1 behind the HMG-1.

LPS, IL-1 are injected in the mouse experiment confirmation or after TNF-8 hour, mononuclear macrophage begins to secrete HMGB1, and serum HMGB1 concentration is kept higher level in 24 h subsequently, and HMGB1 antibody can improve the endotoxemia that LPS causes; Conversely, HMGB1 also can stimulate some proinflammatory factor of mononuclear macrophage secretion, like TNF-, IL-1, II-6, IL-8 etc.Endotoxin shock appearance symptom appears in injection HMGB1 mouse.Human detection finds that also sepsis patient serum HMGB1 level raises, and the degree that raises is relevant with infection seriousness.

When necrocytosis or when impaired, the HMGB1 in the nuclear can be discharged into outside the born of the same parents, causes mononuclear macrophage secretion proinflammatory factor; And proinflammatory factor promotes the secretion of HMGB1 conversely, and positive feedback loop has just formed like this.In the later stage of Inflammatory response, this positive feedback effect has played considerable effect to keeping of Inflammatory response.In recent research, find function and the systemic inflammatory response syndrome of HMGB1, acute lung injury, rheumatoid arthritis, the infraction that mellitus and internal organs ischemic cause is closely related.

Therefore, the material that needs a kind of effective inhibition HMGB1 to exercise short inflammatory factor function is with the treatment inflammation related disease.

Summary of the invention

An object of the present invention is to provide the monoclonal antibody that the secretion specificity combines people's high mobility group protein B 1.

Antibody provided by the present invention can be following 1), 2), 3) or 4) described antibody:

1) a kind of antibody, its variable region of heavy chain have the amino acid residue sequence shown in the sequence 1 in the sequence table, and its variable region of light chain has the amino acid residue sequence shown in the sequence 2 in the sequence table.

2) a kind of antibody, its light chain have the amino acid residue sequence shown in the sequence 8 in the sequence table, and heavy chain has the amino acid residue sequence shown in the sequence 9 in the sequence table.This antibody behaviour-mouse chimeric antibody.

3) a kind of antibody, its amino acid residue sequence is shown in sequence in the sequence table 3.This antibody is single-chain antibody.

4) monoclonal antibody that obtains by the anti-people HMGB1 of hybridoma cell strain hybridoma 3B1 CGMCC No.2907 secretion.

The Fab fragment of being secreted the monoclonal antibody that obtains by the anti-people HMGB1 of hybridoma cell strain hybridoma 3B1 CGMCC No.2907 also belongs to protection scope of the present invention.

Above-mentioned 1) encoding sox of the encoding sox of the variable region of heavy chain of antibody and variable region of light chain also belongs to protection scope of the present invention described in.

Above-mentioned 2) encoding sox of the heavy chain of antibody and light chain also belongs to protection scope of the present invention described in.The light chain encoding sox of said antibody specifically has the nucleotide sequence of sequence 6 in the sequence table, and the heavy chain encoding sox specifically has the nucleotide sequence of sequence 7 in the sequence table.

Above-mentioned 3) encoding sox of antibody also belongs to protection scope of the present invention described in.

Above-mentioned arbitrary said antibody is that application in the medicine of target spot also belongs to protection scope of the present invention with people's high mobility group protein B 1 in preparation.

Said is that the medicine of target spot can be to be used to treat the medicine of the inflammatory reaction of being participated in by people's high mobility group protein B 1, the medicine that is used to treat the medicine of the autoimmune disorder of being participated in by people's high mobility group protein B 1 or is used to treat the organ damage property disease of being participated in by people's high mobility group protein B 1 with people's high mobility group protein B 1.

Said inflammatory reaction is septicemia, systemic inflammatory response syndrome or acute lung injury; Said autoimmune disorder is rheumatoid arthritis, mellitus or lupus erythematosus; Said organ damage property disease is myocardial infarction, cerebral infarction, medicine property or viral liver injury.

The application of above-mentioned arbitrary said antibody in surveyor's high mobility group protein B 1.

Another object of the present invention provides a kind of hybridoma cell line that specificity combines the monoclonal antibody of people's high mobility group protein B 1 of secreting.

This hybridoma cell line is the anti-people HMGB1 of murine hybridoma hybridoma 3B1; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 27th, 2009 and (be called for short CGMCC; Address: Datun Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.2907.

The dissociation constant of experiment proof hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody, the single-chain antibody 3B1scFV that is derived from this monoclonal antibody, people-mouse chimeric antibody ch-3B1 or Fab fragment and people HMGB1 is respectively 7.8nM; 100nM; 7.8nM, 40nM.The IL-6 that hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody, the single-chain antibody 3B1scFV that is derived from this monoclonal antibody, people-mouse chimeric antibody ch-3B1 or Fab fragment can be blocked people HMGB1 stimulates the Raw264.7 cell to produce raises, and in LPS inductive mouse septicemia model, has provide protection.

Said monoclonal antibody and verivate thereof can high-affinity specific recognition high mobility group protein B 1s, and its BA that neutralizes, and can be used to treat septicemia, and systemic inflammatory response is combined and inflammatory reactions such as disease or acute lung injury; Can be used to treat autoimmune disorders such as rheumatoid arthritis, mellitus or lupus erythematosus.The level that also can be used for Clinical Laboratory HMGB1.

Description of drawings

Fig. 1 detects the dissociation curve (ordinate zou is the absorbance value after the ELISA colour developing) of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody and people HMGB1 for ELISA

Fig. 2 is that the binding site of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody on HMGB1 identified.

Fig. 3 is that hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody specificity is identified.

Fig. 4 is that hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody suppresses the IL-6mRNA rise that people HMGB1 stimulates the Raw264.7 cell to produce.

Fig. 5 is the survival of the injection LPS and the mouse of injection LPS and hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody.

Embodiment

Following experimental technique if no special instructions, is ordinary method.

Employed reagent etc. like no specified otherwise, all can be bought from commercial sources among the following embodiment.

The MONOCLONAL ANTIBODIES SPECIFIC FOR and the evaluation of embodiment 1, murine hybridoma CGMCC No.2907 and excretory resisting high mobility group protein B 1 (HMGB1) thereof

One, the preparation of hybridoma cell strain murine hybridoma CGMCC No.2907

1, antigenic preparation:

People HMGB1 and tubercule bacillus (Mycobacterium tuberculosis; MT) acquisition of the fusion rotein (HMGB1-MT) of the little peptide of Psts-1 Argine Monohydrochloride 326-344 (DQVHFQPLPPAVVKLSDAL): hold 181 amino acid whose cDNA and little peptide through the PCR method reorganization coding human HMGB1N, obtain fusion gene HMGB1-MT; (Novagen, USA), clone's plasmid increases in DH5 α, again via expressing among the BL21, obtains fusion rotein HMGB1-MT with fusion gene cloning to the PET-41a carrier of having deleted the GST coding region.Have 6 * hisTag in the fusion rotein, through the Ni-NTA affinitive layer purification.

2, immunity:

The fusion rotein that obtains with step 1 is as antigen immune NZB/W F1 mouse; Immunity is three times altogether: 10 microgram HMGB1-MT add the subcutaneous immunity of complete freund's adjuvant (immunity for the first time), and 5 microgram HMGB1-MT add the subcutaneous immunity of incomplete Freund's adjuvant (immunity for the second time) after 14 days.5 microgram HMGB1-MT add the subcutaneous immunity for the third time of incomplete Freund's adjuvant after 14 days; NZB/W F1 mouse after 3 days is got immunocyte and rat bone marrow tumour SP2/0 cell in the spleen with 5: 1 mixed, merges with polyoxyethylene glycol, obtains hybridoma after the HAT screening.

3, hybridoma screening

The preparation of GST-HMGB1 recombinant protein: the coding region (sequence is shown in sequence in the sequence table 11) of people HMGB1 is cloned into PET-41a carrier (Novagen; USA) EcoR I and HindIII restriction enzyme site; Be converted into DH5 α, resistance screening obtains positive colony, extracts the plasmid of positive colony; Order-checking, the sequence of HMGB1 and direction of insertion are correct in the plasmid as a result; Change positive plasmid over to e. coli bl21,1mM IPTG abduction delivering; Purifying: have GST Tag in the fusion rotein, through the gsh affinitive layer purification, concrete steps are; With the centrifugal collection of bacterium liquid 5000rpm10min after inducing, abandon supernatant, with PBS that thalline is resuspended; Ultrasonication, the centrifugal 10min of 5000rpm abandons deposition; Slowly through gsh chromatography column (Sigma), the fusion rotein that will hang on the gsh chromatography column with the gsh elutriant elutes, and obtains the GST-HMGB1 recombinant protein with supernatant.

The secretion specificity combines the hybridoma of HMGB1 antibody to detect: with GST-HMGB1 recombinant protein (10 mcg/ml) as the coating antigen wrapper sheet; Hybridoma supernatant (carrying out gradient dilution) is one anti-, and HRP link coupled goat anti-mouse igg polyclonal antibody (R&D Systems) is that the two anti-ELISA that carry out detect.

Subclone: repeatedly clone the hybridoma of secreting specificity antibody with restricted dilution method, obtain the anti-people HMGB1 of hybridoma cell strain murine hybridoma hybridoma 3B1 at last.This cell strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 27th, 2009 and (is called for short CGMCC; Address: Datun Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.2907.

4, MONOCLONAL ANTIBODIES SPECIFIC FOR

Extracorporeal culturing method:

The hybridoma CGMCC No.2907 that has set up is placed cell culture medium, place 37 ℃ and 5%CO ₂Cultivate in the incubator, every separated 2d changes cell culture fluid one time, treats that cell concn is greater than 10 ⁵Individual/as to stop to change liquid during ml, it is all dead to continue to cultivate cell.1500rpm, centrifugal 10 minutes, collect culture supernatant, supernatant contains high-caliber monoclonal antibody, and-20 ℃ of preservations are subsequent use.Said cell culture medium is for to add foetal calf serum in the DMEM substratum, making the final concentration of foetal calf serum in cell culture medium is 20% (volumn concentration), and the pH of said cell culture medium is 7.4.

Purifying: the supernatant that vitro culture is obtained carries out following purifying; Above-mentioned supernatant is slowly passed through albumin A/G affinity column (Pierce); The target protein of using 0.1M Glycine/HCl (pH2.5) will hang on the chromatography column again elutes, and obtains the albumen behind the purifying.

5, the Performance Detection of hybridoma CGMCC No.2907 excretory antibody

(1) ELISA detects the dissociation curve of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody and HMGB1.

Experimental technique: (preparation method sees that step 1) (2ug/ml) is as envelope antigen with HMGB1-MT; Anti-(with 160ug/ml is initial concentration to the hybridoma CGMCC No.2907 excretory monoclonal antibody of step 4 preparation purifying as one; Two times two times are diluted), goat anti-mouse igg-HRP (R&D) (1: 2000) is that the two anti-ELISA that carry out measure.

3 repetitions are established in experiment, and the result is as shown in Figure 1, and 50% corresponding AC of ELISA full-scale reading is the dissociation constant of antibody.The dissociation constant that shows hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody and HMGB1 is 7.8nM.The X-coordinate of Fig. 1 is the concentration of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody.

(2) antibody subtype identification experiment: with GST-HMGB1 recombinant protein (10 mcg/ml) wrapper sheet, the hybridoma supernatant is one anti-, HRP link coupled rat anti-mouse IgG1, and IgG2a, or IgG2b monoclonal antibody (BD Pharmingen) is that the two anti-ELISA that carry out detect.Experiment proof hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody is the IgG2b hypotype.

(3) antibody combining site is identified: utilize GST-A box, GST-B box and GST-A+B Box wrapper sheet respectively; Experimental technique: with GST-A box, GST-B box and GST-A+B Box respectively with 2ug/ml as the envelope antigen wrapper sheet; The hybridoma CGMCC No.2907 excretory monoclonal antibody of step 4 preparation is anti-as one, and goat anti-mouse igg-HRP (R&D) (1: 2000) is that the two anti-ELISA that carry out measure.

The preparation process of GST-A box: the coding region (sequence is shown in sequence in the sequence table 14) of people HMGB1 A box is cloned into PET-41a carrier (Novagen; USA) BamHI and HindIII restriction enzyme site; Be converted into DH5 α, resistance screening obtains positive colony, extracts the plasmid of positive colony; Order-checking, the sequence of HMGB1 and direction of insertion are correct in the plasmid as a result; Change positive plasmid over to e. coli bl21,1mM IPTG abduction delivering; Purifying: have GST Tag in the fusion rotein, through the gsh affinitive layer purification, concrete steps are; With the centrifugal collection of bacterium liquid 5000rpm 10min after inducing, abandon supernatant, with PBS that thalline is resuspended; Ultrasonication, the centrifugal 10min of 5000rpm abandons deposition; Slowly through gsh chromatography column (Sigma), the fusion rotein that will hang on the gsh chromatography column with the gsh elutriant elutes, and obtains GST-A box recombinant protein with supernatant.

The preparation process of GST-B box: the coding region (sequence is shown in sequence in the sequence table 15) of people HMGB1 B box is cloned into PET-41a carrier (Novagen; USA) BamHI and HindIII restriction enzyme site; Be converted into DH5 α, resistance screening obtains positive colony, extracts the plasmid of positive colony; Order-checking, the sequence of HMGB1 and direction of insertion are correct in the plasmid as a result; Change positive plasmid over to e. coli bl21,1mM IPTG abduction delivering; Purifying: have GST Tag in the fusion rotein, through the gsh affinitive layer purification, concrete steps are; With the centrifugal collection of bacterium liquid 5000rpm 10min after inducing, abandon supernatant, with PBS that thalline is resuspended; Ultrasonication, the centrifugal 10min of 5000rpm abandons deposition; Slowly through gsh chromatography column (Sigma), the fusion rotein that will hang on the gsh chromatography column with the gsh elutriant elutes, and obtains GST-B box recombinant protein with supernatant.

The preparation process of GST-A+B Box: the coding region (sequence is shown in sequence in the sequence table 11) of people HMGB1 A+Bbox is cloned into PET-41a carrier (Novagen; USA) BamH I and HindIII restriction enzyme site; Be converted into DH5 α, resistance screening obtains positive colony, extracts the plasmid of positive colony; Order-checking, the sequence of HMGB1 and direction of insertion are correct in the plasmid as a result; Change positive plasmid over to e. coli bl21,1mM IPTG abduction delivering; Purifying: have GST Tag in the fusion rotein, through the gsh affinitive layer purification, concrete steps are; With the centrifugal collection of bacterium liquid 5000rpm10min after inducing, abandon supernatant, with PBS that thalline is resuspended; Ultrasonication, the centrifugal 10min of 5000rpm abandons deposition; Slowly through gsh chromatography column (Sigma), the fusion rotein that will hang on the gsh chromatography column with the gsh elutriant elutes, and obtains GST-A+B box recombinant protein with supernatant.

3 repetitions are established in experiment, and the result is as shown in Figure 2, and 3B1 only combines HMGB1 intermediary A Box.

(4) antibodies specific is identified: with the lysate of SDS-PAGE electrophoretic separation human PBMC or Hela cell, carry out western with biotin labeled antibody and detect, Streptavidin-HRP is two anti-, and ECL develops the color.

The human PBMC separates to obtain (blood is provided by the Beijing Red Cross Blood Center) from people's whole blood with the Ficoll density gradient centrifugation; The Hela cell is available from the consonance cell centre, and catalog number is CCC0011.

Streptavidin-HRP is available from middle China fir Golden Bridge, and catalog number is ZB-2404.

Cell lysis buffer solution: available from the green skies, Shanghai Bioisystech Co., Ltd.

The preparation method of the lysate of human PBMC or Hela cell: human PBMC or Hela cell are washed 2-3 time with PBS; Centrifugal collecting cell; Add 100ul cell lysis buffer solution ratio in 500,000 cells and add, add PMSF simultaneously, 4 degree cracking are after 30 minutes; Centrifugal 15 minutes of 12000rpm4 degree is got supernatant and is got final product.

The western detection method: the supernatant that obtains in the lysis is through the 10%SDS-PAGE electrophoresis; Seal (room temperature 2 hours) with 10% skimmed milk-TBS after changeing film; Wash 3 times with TBS-T, (1: the 1000TBS dilution) room temperature reaction is 1 hour, washes 3 times with TBS-T again for same 3B1-biotin; With streptavidin-HRP (1: 8000) room temperature reaction 1 hour, wash 3 times post-exposure with TBS-T.

3 repetitions are established in experiment, and the result is as shown in Figure 3.(CTL representes TBS among Fig. 3) shows that 3B1 antibody can combine with endogenic HMGB1 in the cell pyrolysis liquid (approximately 30kd band) specificity.

The TBS prescription: Tris-HCI damping fluid (0.5M pH7.6) 100ml, NaCI 8.5 ~ 9g (0.15mol/L), distilled water adds to 1000ml.

6, the biological activity of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody is identified

The Raw264.7 cell inoculation in cell culture medium, to wherein adding different stimulator respectively, was cultivated 4 hours; Detect IL-6mRNA level in the cell with real-time RT-PCR, the primer sequence is 5 ' TGG GAAATC GTG GAA ATG AG 3 ', 5 ' CTC TGA AGG ACT CTG GCT TTG 3 '.3 repetitions are established in experiment.

Cell culture medium is formed: RPMI 1640 substratum that added 10% foetal calf serum.

The Raw264.7 cell is available from the consonance cell centre, and catalog number is CCC0146.

The preparation method of GST-HMGB1 recombinant protein: see step 3

Following processing is established in each experiment:

Handle 1: do not add any stimulator;

Handle 2: only add the GST-HMGB1 recombinant protein, its final concentration in culture system is 1ug/ml;

Handle 3: only add 3B1 monoclonal antibody (its final concentration in culture system is 50ug/ml);

Handle 4: the hybridoma CGMCC No.2907 excretory monoclonal antibody (its final concentration in culture system is 50ug/ml) that adds the preparation of GST-HMGB1 recombinant protein (its final concentration in culture system is 1ug/ml) and step 4; Concrete grammar is earlier GST-HMGB1 recombinant protein and hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody to be mixed, and stimulates the Raw264.7 cell again.

Experimental result is as shown in Figure 4, and (1 expression handles 1; 2 expressions handle 2; 3 expressions are handled 3,4 expressions and are handled 4), show that 1ug/ml HMGB-1 can significantly raise the expression level of IL-6mRNA (handling 2); The hybridoma CGMCC No.2907 excretory monoclonal antibody of 50ug/ml can in active with HMGB-1, significantly reduce 1ug/ml HMGB-1 inductive IL-6mRNA and raise (handling 4).The IL-6mRNA that hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody can be blocked people HMGB1 stimulates the Raw264.7 cell to produce raises.

7, the application of anti-HMGB1 antibody in the septicemia mouse model

The C57BL/6 female mice is available from Beijing Vital River Experimental Animals Technology Co., Ltd..LPS is available from Sigma, and catalog number is L2880.

8 age in week 16 of C57BL/6 female mices, heavy 19.5g--20.5g, be divided into 2 groups by the body weight coupling: PBS organizes (LPS+PBS) and antibody group (LPS+3B1).Before the experiment in SPF level Animal House with cage 48 hours, ad lib and water.Inject fasting in preceding 2 hours.PBS group and antibody group mouse give LPS (Sigma 0111:B4) 22.5mg/kg according to body weight.The antibody group gives the hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody 5mg/kg of step 4 preparation after giving LPS, with aseptic PBS damping fluid antibody dilution is arrived final volume 200ul.The PBS group gives PBS damping fluid, 200ul/kg after giving LPS.Intraperitoneal injection.Two component cages are fed, and feeding conditions is identical.Situation after the observation administration.3 repetitions are established in experiment.Two groups of mouse after back 12 hours, are observed Non Apparent Abnormality in injection.But behind 20hr, similar trembling all occur, chaeta is upright, stimulates insensitive performance to external world.After 24 hours, the LPS group begins that dead mouse is arranged; Each treated animal survival changes sees Fig. 5.Explain that hybridoma cell strain 3B1CGMCC No.2907 excretory monoclonal antibody has provide protection in LPS inductive mouse septicemia model.

The preparation of embodiment 2, anti-HMGB1 people-mouse chimeric antibody ch-3B1

Murine antibody can produce immunological rejection in human body, therefore can only be used for the treatment of acute disease, in case produce the anti-antibody to murine antibody in the human body, will lose efficacy as the murine antibody of medicine.In order to overcome this shortcoming, the present invention has carried out humanization to murine antibody, has prepared people-mouse chimeric antibody ch-3B1.The variable region sequences of this antibody is from murine antibody-hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody, and the constant region sequence is from the human IgG1.This antibody has kept the antigen-binding specificity of mouse monoclonal antibody, has reduced inductive immunological rejection in human body simultaneously.

The step of preparation chimeric antibody is following:

Separation and purification mRNA from hybridoma cell strain 3B1 CGMCC No.2907 is with the synthetic first chain cDNA of oligo-dT.With PCR method increase respectively light chain of antibody and variable region of heavy chain, the light chain primer is 5 ' GAY ATT GTG MTSACM CAR WCT MCA 3 ' and 5 ' CTC CAG ATG TTA ACT GCT CAC 3 '; The heavy chain primer is: 5 ' ATGSAR GTN MAG CTG SAG SAG TC 3 ' and 5 ' GGT CAA GGT CAC TGG CTC AGG3 '.Wherein, R=G or A, Y=T or C, M=A or C, S=G or C, W=T or A, N=G or A or C or T.The PCR product is cloned into respectively in the T carrier checks order.The result shows the nucleotide sequence that the encoding sox of the variable region of heavy chain of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody has sequence 4 in the sequence table, and coding has the variable region of heavy chain of the amino acid residue sequence of sequence 1 in the sequence table; The nucleotide sequence that the encoding sox of the variable region of light chain of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody has sequence 5 in the sequence table, coding has the variable region of light chain of the amino acid residue sequence of sequence 2 in the sequence table.

The preparation method of people's light chain and heavy chain constant region gene: separation and purification mRNA from the human PBMC; With the synthetic first chain cDNA of oligo-dT; With PCR method increase respectively people's light chain and heavy chain constant region gene; The light chain primer be 5 ' ttccat act cca gcg ctg cac cat ctg tct tca tct tcc cg3 ' and; 5 ' cct cac tct agagtc gcg gcc gcc taa cac tct ccc ctg ttg aag ctc ttt g 3 ', heavy chain primer are 5 ' gcgtcg acc aag ggc cca tcg gtc ttc c 3 ' and 5 ' acc ctc act cta gag tcg cgg ccgctc att tac ccg gag aca ggg aga ggc t 3 '.

Being connected of the chain variable region gene of monoclonal antibody and people's light chain constant region gene: the one section sequence that earlier 3 ' end of chain variable region gene is added people's light chain constant region gene 5 ' end with PCR method; Primer is 5 ' gct gct gctgtg gtt ccc cgg ctc gcg atg cga tgt ttt gat gac cca aac tcc act ct 3 ' and 5 ' gac aga tgg tgc agc cac agt ccg ttt gat ttc cag ctt g3 '; Be that template is carried out the PCR reaction with chain variable region gene and the people's light chain constant region gene that has one section people's light chain constant region gene again, primer is 5 ' gct gct gct gtg gtt ccc cgg ctc gcg atg cga tgt ttt gat gac cca aac tcc actct 3 ' and 5 ' cct cac tct aga gtc gcg gcc gcc taa cac tct ccc ctg ttg aag ctcttt g 3 '.Hold the one section sequence that has had people's light chain constant region gene 5 ' end because of 3 ' of chain variable region gene, therefore at the annealing stage of PCR, two fragment genes can connect voluntarily.

Being connected of the heavy chain variable region gene of monoclonal antibody and people's heavy chain constant region gene: the one section sequence that earlier 3 ' end of heavy chain variable region gene is added people's heavy chain constant region gene 5 ' end with PCR method; Primer is 5 ' ttt tcttgt cgc gat ttt aaa agg tgt cca gtg cca ggt cca act gca gca gcc t 3 ' and 5 ' ggc cct tgg tcg acg ctg agg aga ctg tga gag tgg tgc c3 '; Be that template is carried out the PCR reaction with heavy chain variable region gene and the people's heavy chain constant region gene that has one section people's heavy chain constant region gene again, primer is 5 ' ttt tct tgt cgc gat ttt aaa agg tgt cca gtg cca ggt cca act gca gca gcc t 3 ' and 5 ' acc ctc act cta gag tcg cgg ccg ctc att tac ccg gag aca ggg aga ggc t3 '.Hold the one section sequence that has had people's heavy chain constant region gene 5 ' end because of 3 ' of heavy chain variable region gene, therefore at the annealing stage of PCR, two fragment genes can connect voluntarily.

The light chain of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody is connected with heavy chain constant region gene with people's light chain respectively with heavy chain variable region gene; Make up the fusion gene of codified chimeric antibody ch-3B1; The encoding sox of this chimeric antibody light chain has the nucleotide sequence of sequence 6 in the sequence table; Its encoding sequence is from 5 of sequence 6 ' the 1st-657 Nucleotide of end, and coding has the ch-3B1 light chain of the amino acid residue sequence of sequence 8 in the sequence table; The encoding sox of this chimeric antibody heavy chain has the nucleotide sequence of sequence 7 in the sequence table, and its encoding sequence is from 5 of sequence 7 ' the 1st-1341 Nucleotide of end, and coding has the ch-3B1 heavy chain of the amino acid residue sequence of sequence 9 in the sequence table.

The structure of chimeric antibody light chain expression vector pCI-gpt-3B1L: the encoding sox (shown in the sequence 5) of this chimeric antibody light chain is inserted into selective mark (guanine phosphoribosyl transferase; Gpt) and gene expression regulation district (CMV promotor; Terminator) in the Nru I of expression vector pCI-gpt and the Afe I site, obtains this chimeric antibody light chain expression vector pCI-gpt-3B1L.

The construction process of expression vector pCI-gpt (is that masterplate makes up with promega carrier pCI): separation and purification mRNA from Hela cell (available from the consonance cell centre, catalog number is CCC0011), with the synthetic first chain cDNA of oligo-dT.Amplify gpt (the gpt gene order is seen sequence 12 in the sequence table) with PCR method, primer is 5 ' ccgtcg cga agc gct atg agc gaa aaa tac atc gtc acc tgg gac3 ' and 5 ' tta gcg accgga gat tgg cgg gac gaa tac 3 '.Gpt is inserted HindIII and the BamHI site of pCI.

The structure of chimeric antibody heavy chain expression carrier pCI-DHFR-3B1H: the encoding sox (shown in the sequence 4) of this chimeric antibody heavy chain is inserted into selective mark (Tetrahydrofolate dehydrogenase DHFR) and gene expression regulation district (CMV promotor; Terminator) the Nru I of expression vector pCI-DHFR and NotI site obtain this chimeric antibody heavy chain expression carrier pCI-DHFR-3B1H.

The construction process of expression vector pCI-DHFR (is that masterplate makes up with promega carrier pCI): separation and purification mRNA from Hela cell (available from the consonance cell centre, catalog number is CCC0011), with the synthetic first chain cDNA of oligo-dT.Amplify DHFR (the DHFR gene order is seen sequence 13 in the sequence table) with PCR method, primer is 5 ' ccg gtc gcg agc ggc cgc atg gtt cga cca ttg aac tgc atc gtc gcc3 ' and 5 ' aagttt gaa gtc tac gag aag aaa gac taa 3 '.DHFR is inserted HindIII and the BamHI site of pCI.

The expression vector pCI-gpt-3B1L and the pCI-DHFR-3B1H that will contain chimeric antibody gene with the method for electrotransfection together import among the mammalian cell NS/0 (ECACC purchase).(Mycophenolate) screens transformant in the substratum that contains xanthine (Xanthine) with mycophenlate mofetil, obtains the cell strain of stable transfection, and note is made NS/3B1.

The cultural method of cell NS/3E8: with RPMI 1640 culture medium culturing that contain 10% foetal calf serum.

Identify the secretion of antibody with ELISA according to the method for embodiment 1.

3 repetitions are established in experiment.

The result shows that the ch-3B1 antibody that obtains has kept the specificity and the avidity of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody, and the dissociation constant of ch-3B1 antibody and HMGB1 is 7.8nM.

The preparation of embodiment 3, anti-HMGB1 single-chain antibody 3B1 scFV

PCR method is the light chain and the heavy chain variable region gene of amplified hybridization tumor cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody respectively; With PCR method heavy chain and variable region of light chain are coupled together with the fifteen amino acid fragment that is rich in glycocoll and Serine then, obtain the coding gene sequence (sequence 10) of anti-HMGB1 single-chain antibody 3B1 scFV.The coding gene sequence of 3B1 scFV is cloned into expression vector pET-26b, and (clone's plasmid increases in DH5 α, again via expressing among the BL21 for Novagen, BamH I USA) and Xho I site.Expression condition is for to induce 2 hours with 1mM IPTG (newly creating development in science and technology ltd available from Beijing good friend).Have 6 * his Tag in the fusion rotein, through the Ni-NTA affinitive layer purification.The amino acid residue sequence that the 3B1 scFV that expresses has sequence 3 in the sequence table.

Method according to embodiment 1 is identified anti-HMGB1 single-chain antibody 3B1scFV with ELISA, and 3 repetitions are established in experiment.The result shows that anti-HMGB1 single-chain antibody 3B1scFV has kept the specificity and the avidity of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody, and the dissociation constant of anti-HMGB1 single-chain antibody 3B1scFV and HMGB1 is 100nM.

The preparation of the Fab fragment 3B1Fab of embodiment 4, anti-HMGB1

Utilize ImmunoPure Fab to prepare the immobilized papain digestion hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody in the test kit (Pierce), full length antibody is degraded into Fab and Fc fragment.Product behind the enzymolysis obtains the antibody fragment of Fab with the immobilization albumin A column purification that provides in the test kit.Identify the Fab fragment 3B1Fab of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody with ELISA according to the method for embodiment 1.3 repetitions are established in experiment.The result shows that the Fab fragment 3B1Fab of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody has kept the specificity and the avidity of hybridoma cell strain 3B1 CGMCC No.2907 excretory monoclonal antibody, and the dissociation constant of anti-HMGB1 single-chain antibody 3B1Fab and HMGB1 is 40nM.

Sequence table

< 110>Institute of Biophysics, Academia Sinica

< 120>monoclonal antibody of resisting high mobility group protein B-1 and application thereof

<160>15

<210>1

<211>115

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>1

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Arg Gly Tyr Tyr Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val

115

<210>2

<211>113

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>2

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ala His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210>3

<211>245

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>3

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Arg Gly Tyr Tyr Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser Gly Ser Gly Ser Ser Gly Ser Gly Ser Ser Gly

115 120 125

Ser Gly Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro

130 135 140

Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

145 150 155 160

Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys

165 170 175

Ala Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr

210 215 220

Cys Phe Gln Gly Ala His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210>4

<211>345

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>4

caggtccaac tgcagcagcc tggggctgag ctggtgaagc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgaatg gattggtaat attgaccctt ctgatagtga aactcactac 180

aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca acagtctgac atctgaggac tctgcggtct attactgtgc aaaaaggggg 300

tactacggct acgactactg gggccaaggc accactctca cagtc 345

<210>5

<211>339

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>5

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120

tacctgcaga aagcaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggtgc acatgttcct 300

cggacgttcg gtggaggcac caagctggaa atcaaacgg 339

<210>6

<211>657

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>6

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120

tacctgcaga aagcaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggtgc acatgttcct 300

cggacgttcg gtggaggcac caagctggaa atcaaacgga ctgtggctgc accatctgtc 360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600

gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt 657

<210>7

<211>1341

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>7

caggtccaac tgcagcagcc tggggctgag ctggtgaagc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgaatg gattggtaat attgaccctt ctgatagtga aactcactac 180

aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca acagtctgac atctgaggac tctgcggtct attactgtgc aaaaaggggg 300

tactacggct acgactactg gggccaaggc accactctca cagtctcctc agcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag ggtctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccgggtaa a 1341

<210>8

<211>219

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>8

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ala His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>9

<211>447

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>9

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Arg Gly Tyr Tyr Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Gly Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>10

<211>735

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>10

caggtccaac tgcagcagcc tggggctgag ctggtgaagc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgaatg gattggtaat attgaccctt ctgatagtga aactcactac 180

aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca acagtctgac atctgaggac tctgcggtct attactgtgc aaaaaggggg 300

tactacggct acgactactg gggccaaggc accactctca cagtctcctc aggctctggc 360

tcttctggct ctggctcttc tggctctggc tcttctgatg ttttgatgac ccaaactcca 420

ctctccctgc ctgtcagtct tggagatcaa gcctccatct cttgcagatc tagtcagagc 480

attgtacata gtaatggaaa cacctattta gaatggtacc tgcagaaagc aggccagtct 540

ccaaagctcc tgatctacaa agtttccaac cgattttctg gggtcccaga caggttcagt 600

ggcagtggat cagggacaga tttcacactc aagatcagca gagtggaggc tgaggatctg 660

ggagtttatt actgctttca aggtgcacat gttcctcgga cgttcggtgg aggcaccaag 720

ctggaaatca aacgg 735

<210>11

<211>510

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>11

atgggcaaag gagatcctaa gatgggcaaa ggagatccta agaagccgag aggcaaaatg 60

tcatcatatg cattttttgt gcaaacttgt cgggaggagc ataagaagaa gcacccagat 120

gcttcagtca acttctcaga gttttctaag aagtgctcag agaggtggaa gaccatgtct 180

gctaaagaga aaggaaaatt tgaagatatg gcaaaagcgg acaaggcccg ttatgaaaga 240

gaaatgaaaa cctatatccc tcccaaaggg gagacaaaaa agaagttcaa ggatcccaat 300

gcacccaaga ggcctccttc ggccttcttc ctcttctgct ctgagtatcg cccaaaaatc 360

aaaggagaac atcctggcct gtccattggt gatgttgcga agaaactggg agagatgtgg 420

aataacactg ctgcagatga caagcagcct tatgaaaaga aggctgcgaa gctgaaggaa 480

aaatacgaaa aggatattgc tgcatatcga 510

<210>12

<211>459

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>12

atgagcgaaa aatacatcgt cacctgggac atgttgcaga tccatgcacg taaactcgca 60

agccgactga tgccttctga acaatggaaa ggcattattg ccgtaagccg tggcggtctg 120

gtaccgggtg cgttactggc gcgtgaactg ggtattcgtc atgtcgatac cgtttgtatt 180

tccagctacg atcacgacaa ccagcgcgag cttaaagtgc tgaaacgcgc agaaggcgat 240

ggcgaaggct tcatcgttat tgatgacctg gtggataccg gtggtactgc ggttgcgatt 300

cgtgaaatgt atccaaaagc gcactttgtc accatcttcg caaaaccggc tggtcgtccg 360

ctggttgatg actatgttgt tgatatcccg caagatacct ggattgaaca gccgtgggat 420

atgggcgtcg tattcgtccc gccaatctcc ggtcgctaa 459

<210>13

<211>564

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>13

atggttcgac cattgaactg catcgtcgcc gtgtcccaaa atatggggat tggcaagaac 60

ggagaccgac cctggcctcc gctcaggaac gagttcaagt acttccaaag aatgaccaca 120

acctcttcag tggaaggtaa acagaatctg gtgattatgg gtaggaaaac ctggttctcc 180

attcctgaga agaatcgacc tttaaaggac agaattaata tagttctcag tagagaactc 240

aaagaaccac cacgaggagc tcattttctt gccaaaagtt tggatgatgc cttaagactt 300

attgaacaac cggaattggc aagtaaagta gacatggttt ggatagtcgg aggcagttct 360

gtttaccagg aagccatgaa tcaaccaggc cacctcagac tctttgtgac aaggatcatg 420

caggaatttg aaagtgacac gtttttccca gaaattgatt tggggaaata taaacttctc 480

ccagaatacc caggcgtcct ctctgaggtc caggaggaaa aaggcatcaa gtataagttt 540

gaagtctacg agaagaaaga ctaa 564

<210>14

<211>237

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>14

atgggcaaag gagatcctaa gaagccgaga ggcaaaatgt catcatatgc attttttgtg 60

caaacttgtc gggaggagca taagaagaag cacccagatg cttcagtcaa cttctcagag 120

ttttctaaga agtgctcaga gaggtggaag accatgtctg ctaaagagaa aggaaaattt 180

gaagatatgg caaaagcgga caaggcccgt tatgaaagag aaatgaaaac ctatatc 237

<210>15

<211>207

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>15

cccaagaggc ctccttcggc cttcttcctc ttctgctctg agtatcgccc aaaaatcaaa 60

ggagaacatc ctggcctgtc cattggtgat gttgcgaaga aactgggaga gatgtggaat 120

aacactgctg cagatgacaa gcagccttat gaaaagaagg ctgcgaagct gaaggaaaaa 180

tacgaaaagg atattgctgc atatcga 207

Claims (11)

1. the monoclonal antibody that produces by hybridoma cell strain murine hybridoma CGMCC No.2907 secretion.

2. hybridoma cell strain murine hybridoma, its deposit number is CGMCC No.2907.

3. antibody, the amino acid residue sequence of its variable region of heavy chain is shown in sequence in the sequence table 1, and the amino acid residue sequence of its variable region of light chain is shown in sequence in the sequence table 2.

4. antibody, the amino acid residue sequence of its light chain is shown in sequence in the sequence table 8, and the amino acid residue sequence of heavy chain is shown in sequence in the sequence table 9.

5. antibody, its amino acid residue sequence is shown in sequence in the sequence table 3.

6. secrete the Fab fragment of the monoclonal antibody that produces by hybridoma cell strain murine hybridoma CGMCC No.2907.

7. the encoding sox of the encoding sox of variable region of heavy chain described in the claim 3 and said variable region of light chain.

8. the encoding sox of heavy chain described in the claim 4 and light chain.

9. the encoding sox of the said antibody of claim 3.

10. claim 1,3 or 4 described antibody or the described Fab fragment of claim 6 are the application in the medicine of target spot with people's high mobility group protein B 1 in preparation;

11. claim 1,3 or 4 described antibody or the described Fab fragment of claim 6 prevent and/or treat the application in the LPS inductive mouse septicemia medicine in preparation.

Images