CN104278013B - Anti-human tissue transglutaminase 2 monoclonal antibody and application thereof - Google Patents
Anti-human tissue transglutaminase 2 monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-human tissue transglutaminase 2 monoclonal antibody and application thereof. A rat hybridoma cell strain, particularly rat anti-human tissue transglutaminase 2 (tGM2) hybridoma cell XK-1 (CCTCC NO:C201312), is established. The hybridoma cell secretes an anti-human tissue transglutaminase 2 (tGM2) monoclonal antibody; and the monoclonal antibody protein has a specific inhibition action on tissue transglutaminase 2 (tGM2), is capable of effectively inhibiting enzyme activity of mouse tGM2 and restoring activity of matrix metal proteinases (MMPs), and has the function of treating liver fibrosis.
Description
Technical field
The invention belongs to biological technical field is and in particular to a kind of anti-human tissue T-5398 2（tGM2）Dan Ke
Grand antibody, further relates to a kind of anti-human tissue T-5398 2（tGM2）Monoclonal antibody treatment hepatic fibrosis in mice in
Application.
Background technology
Liver cirrhosis is a kind of common chronic hepatopathy, is the result of liver severe fibrosis, will lead to the progressively funeral of liver function
Lose, its cause of disease includes hepatoviruses infection, alcohol abuse and other factors.Hepatic fibrosis also provide ring for tumor
Border, it can not only activate tumor cell, also can promote the transfer of tumor.National hepatitis stream venerology's survey result shows within 2006
Show, population of China HbsAg carrier rate is 7.18%, and estimation there are about 4.57 hundred million people and once infected hepatitis B virus, its
In 93,000,000 people be HBsAg carrier, the incidence rate of its liver cirrhosis and hepatocarcinoma is very high.Hepatocarcinoma is at present on Cancer in China mortality rate
It is number two.China is every year because the economic loss that chronic hepatopathy and liver cirrhosis cause is estimated as 500,000,000,000 yuans.In the U.S.
About 4 million peoples suffer from chronic hepatitiss, and the mortality rate ranking the 8th of hepatocarcinoma.Every year because chronic hepatopathy and liver cirrhosis cause
Economic loss be estimated as 1,600,000,000 dollars.But what the method for effectively treatment hepatic fibrosis in the world was a lack of.Tight for this
High present situation, in the urgent need to researching and developing a kind of innovative approach of effective treatment hepatic fibrosis.
Hepatic fibrosis originate from hepatocyte epimatrix（ECM）Excessive interconnection, this connection has resulted in liver group
Knit the increase of rigidity and irreversible.In multiple ECM cross-linking enzymes, tTG 2（tGM2）Having been observed that is
One critically important factor, occurs in the pathogenesis of the various tissue fibering including liver cirrhosis.In order to resist
The activity of tGM2, the micromolecular inhibitor with alkylation function has been developed.However, their alkylation effect and tissue
Contact is poor, hampers the treatment use of these micromolecular inhibitors.Our data shows, hepatic fibrosis are that occur
In the state of immunologic tolerance.Matrix metalloproteinase in this case（MMPs）Expression be suppressed, immune surveillance system is subject to
Damage, tTG 2（tGM2）Expression and enzymatic activity increase, ultimately result in fibrous tissue hyperplasia and tumor
Growth.
Existing discovery gives us an enlightenment：If a kind of monoclonal antibody can be prepared, the enzyme of tGM2 can be suppressed
Activity, can activate the cell-mediated cytotoxic effect of antibody-dependant again（ADCC）, recover matrix metalloproteinase in fiber
Change the expression of tissue surface and activity, it will greatly promote and Fibrotic dissolve and disappear.Based on this theory, we are successful
Establish one plant of anti-human tissue T-5398 2（tGM2）Monoclonal antibody.This antibody can not only vitro inhibition people
The enzymatic activity of tGM2, and matrix metalloproteinase enzyme can be recovered in mouse model（MMPs）Expression, reduce hepatic fibrosis degree,
Have shown that the therapeutic efficiency of this antibody.
Content of the invention
The purpose of the present invention is to there are provided a kind of anti-human tissue T-5398 2（tGM2）Monoclonal antibody,
This monoclonal antibody is by rat anti-human tTG 2（tGM2）Hybridoma XK-1 secretes, targeted target
Site is located at people tTG 2（tGM2）Enzyme active sites in, highly protect between multiple species such as mice and people
Keep.This antibody shows the specificity of height, and can effectively suppress tTG 2（tGM2）Enzymatic activity.
A further object of the invention is to there are provided a kind of rat anti-human tTG 2（tGM2）Hybridization
Oncocyte XK-1, this hybridoma can secrete anti-human tissue T-5398 2（tGM2）Monoclonal antibody, this cell is
Deliver to China typical culture collection center on January 14th, 2013 and carry out preservation, Classification And Nomenclature：Rat anti-human's tissue turns paddy ammonia
Amidase 2（tGM2）Hybridoma XK-1, deposit number：CCTCC NO:C201312, address：Wuhan, China Wuhan University.
Last purpose of the present invention is to there are provided a kind of anti-human T-5398 2（tGM2）Monoclonal anti
Application in preparation treatment hepatic fibrosis in mice medicine for the body.
To achieve these goals, the present invention adopts following technical measures：
First, the synthesis of the design of target antigen determinant and polypeptide：
Using literature search and protein amino acid sequence analysis, applicant is in T-5398 2（tGM2）Enzyme activity
Property region be found that one section of highly conserved aminoacid sequence, its sequence be SEQ ID NO:Shown in 1.This sequence is in mice, big
Highly conserved between Mus and the mankind.Applicant has carried out the synthesis of polypeptide (being synthesized by NeoBiolab company of the U.S.) by this sequence.
Polypeptide and carrier protein keyhole hemocyanin（Keyhole Limpet Hemocyanin, abbreviation KLH）It is coupled (by the U.S.
NeoBiolab company synthesizes), remove immune rat as immunogen.
2nd, anti-human tissue T-5398 2（tGM2）The preparation of monoclonal antibody：
The immunity of A, SD rat
The SD rat being 12 weeks as 3 ages of antigen immune with KLH- polypeptide coupling protein.250ug antigen is dissolved in no
In the phosphate buffer of bacterium, and heat in 50 DEG C of water-baths 30 minutes, then mix with Freund's complete adjuvant, make uniformly
Oily milk, and antigen oil milk is injected in the abdominal cavity of rat.After 3 weeks, with same method by antigen and Fu Shi
Freund's incomplete adjuvant mixes, and is injected in the abdominal cavity of rat.After 3 weeks, with same method by antigen and freund 's incomplete adjuvant
Mix, and be injected in the abdominal cavity of rat.The last time 2 weeks after immunity, labeling method is joined by the blood sampling of eyeball periphery and enzyme
To determine the immune success rate of KLH- polypeptide coupling protein.
B, cell fusion
One month after determining immune success rate, make booster immunization.By 200ug KLH- polypeptide coupling protein antigen through tail
Intravenous injection is in positive rats.After 2 days, separately negated immune rat, prepares trophoblastic cell.After 4 days, put to death by immune big
Mus, take out spleen with sterile scissors and tweezers, and with pH7.4,0.1mol/L phosphate buffer washes away blood, existed with aseptic syringe needle
In sterile petri dish, spleen is bundled into porous, lymphocyte is released in the DMEM culture fluid of serum-free, finally uses serum-free
DMEM culture fluid make cell suspending liquid, as immune rat splenocyte parent, standby.Take 2 × 107Individual IR983F is big
Rat bone marrow tumour cell（Purchased from Shanghai Dou Yi biotechnology company）With 108Individual immune rat splenocyte is in 50ml centrifuge tube
In, mix in the DMEM culture fluid of serum-free, 300g is centrifuged 5 minutes, sucks supernatant, is repeated 2 times.Finally flick ttom of pipe,
So that the cell of precipitation is loosened, be incubated in 37 degree of water-baths, and gently Deca 1ml 50%PEG fusion agent in 1 minute（Sigma is public
Department）, gently springing cell 60～90 seconds simultaneously, then Deca 2ml serum-free DMEM culture fluid in 1 minute, with after 2 minutes
The culture fluid of the interior DMEM adding 20 milliliters of serum-frees.Centrifugation 300g, 5min, suck supernatant, then again with containing 20% calf blood
Clear HAT-DMEM culture fluid is diluted to 100ml, makes cell suspending liquid, uniformly point is added on 10 piece of 96 hole with every hole 100ul thin
In born of the same parents' culture plate.Then in 37 degree, cultivate 12-15 days in C02 incubator.Period, replacing HAT cell culture fluid 4 times.After culture
Phase, if culture fluid appearance is faint yellow in culture hole, after observation confirmation is with the presence of cell clone, can be taken off a part of culture fluid
Carry out antibody test.
C, the screening of hybridoma：
It is expressed as the hybridoma cell clone of the positive with the method screening antibodies of enzyme linked immunological labelling.The polypeptide of synthesis is resisted
Former（The polypeptide that non-KLH is coupled）It is dissolved in 0.05M carbonic acid buffer（PH9.6）, concentration is 10ug/ml, is coated in 96 hole polyphenyl
Vinyl plate, 4 DEG C overnight.Use phosphate buffer（PBS）Wash 3 times afterwards, close 96 orifice plates with 0.5%BSA solution, 37 DEG C, 1 is little
When.Hybridoma culture supernatant to be measured adds 96 orifice plates, 37 DEG C, cultivates 1 hour, then uses phosphate buffer（PBS）Washing 3 times,
Add peroxidase（HRP）The two of the anti-rat IgG being coupled resist（GE Healthcare company）, 37 DEG C, cultivate 30 points
Clock.Use phosphate buffer（PBS）+ 0.1% tween（Tween-20）Washing 4 times, with tetramethyl benzidine (TMB) colour developing, through enzyme mark
Instrument quantitative determines, to determine the positive hole producing anti-polypeptide monoclonal antibody.
It is defined as producing the positive hole cell of anti-tGM2 polypeptide antibody after testing, be first transferred to 24 well culture plate cultures and expand
Increase, reaffirm the hybridoma cell clone of the positive with the method for enzyme linked immunological labelling, then transfer cell is to T25 culture bottle
Culture amplification.Will be frozen for positive hybridoma cell clone, select 5 antibody expression highests clone simultaneously and carry out again gram
Grand, and through comprehensive identification and analysis, finally could obtain the monoclonal hybridoma system of anti-tGM2 polypeptide, thereby is achieved one
The monoclonal hybridoma of the anti-human tGM2 of strain, this cell is delivered in China typical culture collection on January 14th, 2013
The heart carries out preservation, Classification And Nomenclature：Rat anti-human tTG 2（tGM2）Hybridoma XK-1, deposit number：
CCTCC NO:C201312, address：Wuhan, China Wuhan University.
This hybridoma is similar to general hybridoma, belongs to suspended culture cell, can in RPMI 1640,10%
Cultivate in FBS culture medium, cell cycle is about 17 hours.Hybridoma secretes the monoclonal anti of anti-tGM2 in culture medium
Body, antibody concentration can reach 25pg/ml.
D, anti-human tissue T-5398 2（tGM2）The purification preparation of monoclonal antibody：
The monoclonal antibody of anti-human tGM2 can be produced by inducing ascites tumor, and its process is as follows：Big to healthy SD
Mus lumbar injection 1ml norphytane (Pristane), after raising 2 weeks, inoculate 5 × 10 to rat abdominal cavity6Individual hybridoma, raises
Ascites can substantially be produced after 9～11 days, when ascites volume reaches to greatest extent, extract ascites with capillary tube, typically can obtain
50ml about.Ascites is centrifuged 10min through 1000rpm/min, discards upper-layer fat layer, collects middle level ascites, and -80 DEG C of preservations are standby
With.
The the isolating and purifying of the monoclonal antibody of anti-human tGM2：The ascites collected, after the dilution of 4 times of normal saline, the body such as adds
Long-pending saturated ammonium sulfate solution（PH=7.4）Mix homogeneously, 4 DEG C precipitate 1 hour.Then 8000rpm/min is centrifuged 30 minutes.Receive
Collection precipitation, and be redissolved in isopyknic normal saline, add saturated ammonium sulfate solution（pH=7.4）So as to volume accounts for
The 45% of cumulative volume, 4 DEG C precipitate 1 hour.After being centrifuged 30 minutes through 8000rpm/min, precipitation is redissolved in appropriate physiology
In saline, -20 DEG C save backup.
Protein G affinity column (5ml, HiTrap using Amersham Pharmacia Biotech company
Protein G HP) carry out affinity chromatograph.Before loading, with 20mM, the phosphate buffer balance affine layer of protein G of pH=7.6
Analysis post.Draw the sample that 2ml saltouts, slow loading through saturated ammonium sulfate.After sample is completely into chromatographic column, with 20mM, pH=
7.6 phosphate buffer eluting foreign protein, flow velocity is 0.5ml/min.After foreign protein eluting is complete, with 100mM, the lemon of pH=3.2
Lemon acid buffer antibody elution albumen, flow velocity is 1ml/min, and often 1ml collected by pipe.In advance added with 100ul, pH in every collecting pipe
=9.0 1M Tris-HCL buffer, for neutralizing eluent to pH=7.0.The second eluting collected through UV-280nm detection
Peak is the antibody protein wanting purification.Then, through ultrafiltration, concentrate the monoclonal antibody of the anti-tGM2 obtaining final product high concentration.Pure antibody
Degree is more than 96% through SDS-PAGE electroresis appraisal, purity.
3rd, anti-human tissue T-5398 2（tGM2）Monoclonal antibody preparation treatment hepatic fibrosis in mice medicine
In application, its application process is：
A, the foundation of hepatic fibrosis in mice model：
By repeatedly injecting thioacetamide (TAA)（0.2mg/g body weight, biweekly, co-injection 8 weeks）Set up little
Liver cirrhosis model.Fig. 1, double fluorescence immunization colorations show, tGM2 does not almost express in Normal mouse liver is dirty, however,
In fibrosiss liver, the expression of tGM2 substantially increases, and shows with Fibrotic type i collagen bridge phase and deposit.Importantly,
In mice Hepatocirrhosis Model, tGM2 is much like with the distribution situation in people's cirrhosis, liver in the distribution in fibrosiss hepatic tissue（See
Fig. 1）.
B, the therapeutic effect on hepatic fibrosis in mice model for the monoclonal antibody of anti-human T-5398 2：
Mice is by duplicate injection TAA（0.2mg/g body weight, 2 times/week, totally 8 weeks).After 8 weeks, mice significantly loses body
Weight, hepatic fibrosis symptom occurs.Hepatic fibrosis mice is divided into two groups.First group is experimental group（21 mices）, will be injected
The antibody of anti-human tGM2, the dosage of antibody is 0.005 milligram of every mice respectively, 0.02 milligram or 0.1 milligram, every kind of dosage
7 mices of injection.Second group is matched group（7 mices）, normal rat IgG will be injected, dosage is 0.1 milligram.3rd
Organize as blank group（7 mices）, mice is not through the injection of TAA and antibody.The last time two days after TAA injection, pass through
The antibody of the anti-tGM2 of lumbar injection or normal rat IgG.5 days afterwards, then injects a wheel antibody.After 5 days, mice was killed,
Therapeutic effect is evaluated.
Fig. 2A shows Picro-Sirius red（Sirius Red）The result of dyeing, is compared with matched group, and hepatic fibrosis knot exists
Anti- tGM2 Antybody therapy group（0.1 milligram）Substantially reduce.Fig. 2 B shows, markers of fibrosis molecule alpha-smooth muscle actin（α-
SMA）Expression reduce also with the increase of anti-tGM2 antibody dosage.Fig. 2 C shows, in the table of the downtrod MMP9 of matched group
Reach part to recover, but the injection in the antibody of the anti-tGM2 of experimental group is significantly enhanced the expression of MMP9.This with little
Disappearing of liver fibrosis is consistent.Zoopery shows, the antibody protein of anti-human tissue T-5398 is as medicine energy
Effectively suppress the enzymatic activity of tTG, greatly promote the ablation of hepatic fibrosis, hard for treatment people's hepatic fibrosis, liver
Change and provide new approach.
4th, compared with prior art, the present invention has advantages below：
The common drug of domestic treatment hepatic fibrosis includes Colchicine, interferon, ursodesoxycholic acid etc. at present.Colchicum autumnale
Alkali passes through to suppress tubulin polymerization, the collagen secretion of interference cell, stimulates the activity of collagenase, increases the degraded of collagen, comes
Mitigate degree of hepatic fibrosis, but often cause Nausea and vomiting, appetite reduction, diarrhoea, constipation and abdominal distention, have strong toxic and side effects.
Interferon can suppress the activation of hepatic stellate cell, propagation, has the effect of anti-hepatic fibrosis, but can cause heating, and big dose can
Cause reversibility cytopenia, based on leukocyte and thrombocytopenia, erythrocytopenia, the big toxic and side effects of dose are strong.Bearss go
It is not high that oxycholic acid improves degree in long-time medication heptic fibrosis.Abroad, there are the suppression hepatic fibrosis of alkylation function
Micromolecular inhibitor be developed.However, their alkylation effect and contact tissue are poor, hamper these little point
The treatment use of sub- inhibitor.At present, clinically need the new drug of more preferable more safe and effective anti-hepatic fibrosis.
Important function, during people's hepatic fibrosis, has played in people tTG 2 (tGM2).Anti-human tissue transglutamin-ase 9
The monoclonal antibody of enzyme 2, can specifically suppress the enzymatic activity of people tTG 2 (tGM2), as macromolecular drug,
More conventional medicine shows many superiority：（1）It is the targeting of this monoclonal antibody first, as guided missile, can be directionally
Hit target, specifically combine the T-5398 2 (tGM2) on people's fibrosiss hepatic tissue surface, suppress the work of this enzyme
Property, stop the further fibrosiss of liver, meanwhile, this antibody has little combination to other normal human tissue organs, thus malicious
Small side effects；（2）This affinity of antibody is strong, and low dose of antibody just can play its curative effect, reduces the poison to human tissue organ
Side effect；（3）By the cell-mediated cytotoxic effect of antibody-dependant（ADCC）, this antibody can be by matrix metalloproteinase
（MMPs）Gather the surface of fibrosiss liver, directly dissolve Fibrotic extracellular medium, recover elasticity and the function of liver；（4）
As macromolecular drug, the preparation technology of monoclonal antibody is ripe, and technique is convenient, and production cost can be very low, great industry
Change and be worth.
Brief description
Fig. 1 is a kind of double fluorescence method immunostaining schematic diagrams of mice Hepatocirrhosis Model.
Double fluorescence method immunostainings show, markers of fibrosis molecular collagen I and tTG 2 (tGM2)
Coexist in hepatic fibrosis in mice position.
Fig. 2 is a kind of anti-human tissue T-5398 2（tGM2）Monoclonal antibody is on hepatic fibrosis in mice model
Therapeutic effect schematic diagram.
The monoclonal antibody of anti-human tissue T-5398 2, by recovering the activity of matrix metalloproteinase MMP9, disappears
Subtract hepatic fibrosis in mice.（A）Picro-Sirius red（Sirius Red）The result of dyeing shows, hepatic fibrosis are tied in anti-human tGM2
Substantially reduce under the treatment of monoclonal antibody；(B) markers of fibrosis molecule alpha-smooth muscle actin（α-SMA）Expression with
The increase of antibody dosage and reduce；(C) treatment of anti-human tGM2 antibody significantly increases the expression of MMP9.
Fig. 3 is a kind of anti-human tissue T-5398 2（tGM2）Monoclonal antibody is illustrated to the inhibition of enzyme activity of tGM2
Figure.
With people tTG 2（tGM2）Zymophore be target rat monoclonal antibody, in vitro
The activity of its transaminase can effectively be suppressed.（A）The monoclonal antibody of anti-human tissue T-5398 2 restrained effectively
The biotinylated peptide substrate that tGM2 is catalyzed and the crosslinking of poly-D-lysine；（B）Show that the functional structure of people tGM2 is shown
It is intended to and enzyme active sites, and the polypeptide antigen site coming from enzyme active sites.
Fig. 4 is a kind of people tTG 2（tGM2）Expression schematic diagram on the liver sample of patient with liver cirrhosis.
Coloration result shows, people tTG 2（tGM2）Have aobvious at the hepatic fibrosis position of patient with liver cirrhosis
The overexpression of work property.（A）Immunofluorescence dyeing shows, people tGM2 and markers of fibrosis molecule alpha-smooth muscle actin（α-
SMA）Coexist in people's hepatic fibrosis position；（B）Picro-Sirius red（Sirius Red）The result of dyeing；（C）Display cirrhotic tissue
Zhong Ren tTG 2（tGM2）, α-smooth muscle actin（α-SMA）MRNA expression with collagen protein I
Dramatically increase.
Fig. 5 is a kind of immunosuppressant of fibrosiss liver and the schematic diagram of matrix metalloproteinase disappearance
The result of study of hepatic fibrosis in mice model shows, at hepatic fibrosis position, matrix metalloproteinase (MMPs)
Expression is subject to suppression, and immunologic tolerance.(A) after TAA process, the glutamate pyruvate transaminase of normal hepatocytes（ATL）Level increases by 6
Times, but the ATL level of fibrosiss liver does not have significant changes；(B) under conditions of acute injury, compared with fibrosiss liver, normally
The degree of injury that liver is subject to is more serious；(C) compared with normal hepatocytes, the expression of the collagen protein I mRNA of fibrosiss liver increases
4 times；(D) after acute injury, the expression of the matrix metalloproteinase MMP9 of normal hepatocytes dramatically increases, and in fibrosiss
The expression of liver MMP9 does not have significant changes；（E）With（F）After acute injury, the expression of the TNF-α of normal hepatocytes and IL-1 β
Level all dramatically increases, and the expression in fibrosiss liver TNF-α and IL-1 β does not have significant changes.
Specific embodiment
Embodiment 1：Polypeptide design and synthesis
Using literature search and protein amino acid sequence analysis software, applicant is in tTG 2
（tGM2）Enzymatic activity region be found that one section of highly conserved aminoacid sequence, its sequence be SEQ ID NO.1 shown in.This sequence
It is listed in mice, highly conserved between rat and the mankind.Applicant has carried out the synthesis of polypeptide (by the U.S. by this sequence
NeoBiolab company synthesizes).Part of polypeptide and carrier protein keyhole hemocyanin（Keyhole Limpet Hemocyanin,
Abbreviation KLH）It is coupled (being synthesized by NeoBiolab company of the U.S.), remove immune rat as immunogen.
Embodiment 2：Anti-human tissue T-5398 2（tGM2）The preparation of monoclonal antibody
The immunity of A, SD rat
The SD rat being 12 weeks as 3 ages of antigen immune with KLH- polypeptide coupling protein.250ug antigen is dissolved in no
In the phosphate buffer of bacterium, and heat in 50 DEG C of water-baths 30 minutes, then mix with Freund's complete adjuvant, make uniformly
Oily milk, and antigen oil milk is injected in the abdominal cavity of rat.After 3 weeks, with same method by antigen and Fu Shi
Freund's incomplete adjuvant mixes, and is injected in the abdominal cavity of rat.After 3 weeks, with same method by antigen and freund 's incomplete adjuvant
Mix, and be injected in the abdominal cavity of rat.The last time 2 weeks after immunity, labeling method is joined by the blood sampling of eyeball periphery and enzyme
To determine the immune success rate of KLH- polypeptide coupling protein.
B, cell fusion
One month after determining immune success rate, make booster immunization.By 200ug KLH- polypeptide coupling protein antigen through tail
Intravenous injection is in positive rats.After 2 days, separately negated immune rat, prepares trophoblastic cell.After 4 days, put to death by immune big
Mus, take out spleen with sterile scissors and tweezers, and with pH7.4,0.1mol/L phosphate buffer washes away blood, existed with aseptic syringe needle
In sterile petri dish, spleen is bundled into porous, lymphocyte is released in the DMEM culture fluid of serum-free, finally uses serum-free
DMEM culture fluid make cell suspending liquid, as immune rat splenocyte parent, standby.Take 2X107Individual IR983F is big
Rat bone marrow tumour cell（Purchased from Shanghai Dou Yi biotechnology company）With 108Individual immune rat splenocyte is in 50ml centrifuge tube
In, mix in the DMEM culture fluid of serum-free, 300g is centrifuged 5 minutes, sucks supernatant, is repeated 2 times.Finally flick ttom of pipe,
So that the cell of precipitation is loosened, be incubated in 37 degree of water-baths, and gently Deca 1ml 50%PEG fusion agent in 1 minute（Sigma is public
Department）, gently springing cell 60～90 seconds simultaneously, then Deca 2ml serum-free DMEM culture fluid in 1 minute, with after 2 minutes
The culture fluid of the interior DMEM adding 20 milliliters of serum-frees.Centrifugation 300g, 5min, suck supernatant, then again with containing 20% calf blood
Clear HAT-DMEM culture fluid is diluted to 100ml, makes cell suspending liquid, uniformly point is added on 10 piece of 96 hole with every hole 100ul thin
In born of the same parents' culture plate.Then in 37 degree, CO2Cultivate 12-15 days in incubator.Period, replacing HAT cell culture fluid 4 times.After culture
Phase, if culture fluid appearance is faint yellow in culture hole, after observation confirmation is with the presence of cell clone, can be taken off a part of culture fluid
Carry out antibody test.
C, the screening of hybridoma：
It is expressed as the hybridoma cell clone of the positive with the method screening antibodies of enzyme linked immunological labelling.The polypeptide of synthesis is resisted
Former（The polypeptide that non-KLH is coupled）It is dissolved in 0.05M carbonic acid buffer（PH9.6）, concentration is 10ug/ml, is coated in 96 hole polyphenyl
Vinyl plate, 4 DEG C overnight.Use phosphate buffer（PBS）Wash 3 times afterwards, close 96 orifice plates with 0.5%BSA solution, 37 DEG C, 1 is little
When.Hybridoma culture supernatant to be measured adds 96 orifice plates, 37 DEG C, cultivates 1 hour, then uses phosphate buffer（PBS）Washing 3 times,
Add peroxidase（HRP）The two of the anti-rat IgG being coupled resist（GE Healthcare company）, 37 DEG C, cultivate 30 points
Clock.Use phosphate buffer（PBS）+ 0.1% tween（Tween-20）Washing 4 times, with tetramethyl benzidine (TMB) colour developing, through enzyme mark
Instrument quantitative determines, to determine the positive hole producing anti-polypeptide monoclonal antibody.
It is defined as producing the positive hole cell of anti-tGM2 polypeptide antibody after testing, be first transferred to 24 well culture plate cultures and expand
Increase, reaffirm the hybridoma cell clone of the positive with the method for enzyme linked immunological labelling, then transfer cell is to T25 culture bottle
Culture amplification.Will be frozen for positive hybridoma cell clone, select 5 antibody expression highests clone simultaneously and carry out again gram
Grand, and through comprehensive identification and analysis, obtain the monoclonal hybridoma system of one plant of anti-tGM2 polypeptide.This cell is in 2013
January 14 was delivered to China typical culture collection center and carries out preservation, Classification And Nomenclature：Rat anti-human tTG 2
（tGM2）Hybridoma XK-1, deposit number：CCTCC NO:C201312, address：Wuhan, China Wuhan University.
D, the purification preparation of the monoclonal antibody of anti-human tGM2 polypeptide：
The monoclonal antibody of anti-human tGM2 can be produced by inducing ascites tumor, and its process is as follows：Big to healthy SD
Mus lumbar injection 1ml norphytane (Pristane), after raising 2 weeks, inoculate 5x10 to rat abdominal cavity6Individual hybridoma, raises 9
Ascites can substantially be produced after～11 days, when ascites volume reaches to greatest extent, extract ascites with capillary tube, typically can obtain 50ml
Left and right.Ascites is centrifuged 10min through 1000rpm/min, discards upper-layer fat layer, collects middle level ascites, -80 DEG C save backup.
The the isolating and purifying of the monoclonal antibody of anti-human tGM2：The ascites collected, after the dilution of 4 times of normal saline, the body such as adds
Long-pending saturated ammonium sulfate solution（PH=7.4）Mix homogeneously, 4 DEG C precipitate 1 hour.Then 8000rpm/min is centrifuged 30 minutes.Receive
Collection precipitation, and be redissolved in isopyknic normal saline, add saturated ammonium sulfate solution（PH=7.4）So as to volume accounts for
The 45% of cumulative volume, 4 DEG C precipitate 1 hour.After being centrifuged 30 minutes through 8000rpm/min, precipitation is redissolved in appropriate physiology
In saline, -20 DEG C save backup.
Protein G affinity column (5ml, HiTrap using Amersham Pharmacia Biotech company
Protein G HP) carry out affinity chromatograph.Before loading, with 20mM, the phosphate buffer balance affine layer of protein G of pH=7.6
Analysis post.Draw the sample that 2ml saltouts, slow loading through saturated ammonium sulfate.After sample is completely into chromatographic column, with 20mM, pH=
7.6 phosphate buffer eluting foreign protein, flow velocity is 0.5ml/min.After foreign protein eluting is complete, with 100mM, the lemon of pH=3.2
Lemon acid buffer antibody elution albumen, flow velocity is 1ml/min, and often 1ml collected by pipe.In advance added with 100ul, pH in every collecting pipe
=9.0 1M Tris-HCL buffer, for neutralizing eluent to pH=7.0.The second eluting collected through UV-280nm detection
Peak is the antibody protein wanting purification.Then, through ultrafiltration, concentrate the monoclonal antibody of the anti-tGM2 obtaining final product high concentration.Pure antibody
Degree is more than 96% through SDS-PAGE electroresis appraisal, purity.Western Blot experimental result only shows a 80KD positive
Band, illustrates that this antibody can be specifically combined with tGM2.This antibody is rat IgG2b hypotype, and antibody highest dilutes up to 0.5ug/
ml.
Embodiment 3:The identification of the functional activity of anti-human tGM2 monoclonal antibody
In order to detect the activity of monoclonal antibody suppression tGM2, applicant establishes an enzymatic reaction system.At this
In system, from people's I-type collagen one section of polypeptide first by Biotin modificationization (Biotin-LSQQIENI), Ran Hou
It is linked to poly-D-lysine under the catalysis of tGM2（poly-lysine）Upper (Fig. 3). the degree of cross-linking reaction is to pass through
The enzymatic reaction of avidin-HRP mediation is monitoring.Fig. 3 shows, with the increase of the antibody concentration of anti-tGM2, tGM2 turns ammonia
Activity is suppressed significantly.
Embodiment 4：People tTG 2（tGM2）Expression on the liver sample of patient with liver cirrhosis
Using immunofluorescence dyeing method, applicant has made to the liver sample of 12 patient with liver cirrhosis to check.Fig. 4 A ,-
The dyeing of smooth muscle actin（Green）Disclose：Regardless of the cause of disease（HCV, HBV, PBC, or HCC infects）, great majority
Liver cirrhosis sample is clearly present of typical nodular fibrosis bridge（nodular fibrotic bridges）.TGM2 contaminates
Color（Red）Disclose：TGM2 albumen is widely expressed in fibrosiss bridge and about, is shown that tGM2 is tight with Fibrotic
Close correlation.The surrounding of pernicious pseudolobuli there is also the positive fiber bridge of tGM2.Fig. 4 B, Picro-Sirius red（Sirius Red）
Dyeing discloses the fiber section on liver sample, i.e. liver cicatrix.Using qRT-PCR method we also have detected hardening liver
The expression of the mRNA of tGM2.Fig. 4 C shows：I-type collagen and-smooth muscle actin（Two kinds of typical fibrosiss refer to
Mark）MRNA level in-site increased respectively more than 5 times and more than 2 times.The mRNA of tGM2 also significantly increases 2 times.Finally, we detect
The enzymatic activity of extracellular matrix tGM2, finds that the transaminase activity of the tGM2 of hardening liver dramatically increases.
Embodiment 5：The foundation of hepatic fibrosis in mice model
By repeatedly injecting thioacetamide (TAA)（0.2mg/g body weight, biweekly, co-injection 8 weeks）Set up little
Liver cirrhosis model.Fig. 1, double fluorescence immunization colorations show, tGM2 does not almost express in Normal mouse liver is dirty, however,
In fibrosiss liver, the expression of tGM2 substantially increases, and shows with Fibrotic type i collagen bridge phase and deposit.Importantly,
In mice Hepatocirrhosis Model, tGM2 is much like with the distribution situation in people's cirrhosis, liver in the distribution in fibrosiss hepatic tissue（See
Fig. 1）.
Embodiment 6：The immunosuppressant of liver fibrosis and the disappearance of matrix metalloproteinase
The hepatic fibrosis being caused due to acute injury can pass through matrix metalloproteinase（MMPs）Spontaneously remove.So
And, because the liver cirrhosis cicatrix that chronic hepatitiss cause but can not be dissolved in time.This is because the liver of fibrosiss liver is starlike thin
Many matrix metalloproteinases in born of the same parents（MMPs）Gene be silenced change, protease no longer express (Qin and Han,
2010).Since in Fibrotic hepatic stellate cell, the activity of MMPs is for good and all suppressed, then Fibrotic for dissolving
MMPs may be from the lymphocyte of leukocyte or bone marrow derived.The expression of most of MMPs genes is by proinflammatory cytokine
The factor (TNF-α and IL-1 β) induces.It is true that showing, fibrosiss liver is anti-from plasma A LT horizontal regulating liver-QI bleeding
TAA damages challenge（See Fig. 5 A and 5B）, and normal hepatocytes can enter necrosis after damaging challenge through TAA.Acute injury is just
Often liver have expressed substantial amounts of matrix metalloproteinase MMP9, and the liver hardening does not show the expression of MMP9 (see figure
5D). main pro-inflammatory cytokine, TNF-α and IL-1 β, only in the liver of acute injury, expression increases, but in fibre
But do not increase in the liver of dimensionization（See Fig. 5 E and 5F）.Therefore, applicant thinks in the hepatic tissue that liver cirrhosis occurs and exists
Immunosuppressant, and this immunosuppressant protects fibrosed tissue from immune attack, eliminates matrix metal egg
The expression of white enzyme, and promote generation and the transfer of tumor.
Embodiment 7：Therapeutic effect on hepatic fibrosis in mice model for the anti-human tGM2 monoclonal antibody
Mice is by duplicate injection TAA（0.2mg/g body weight, 2 times/week, totally 8 weeks).After 8 weeks, mice significantly loses body
Weight, hepatic fibrosis symptom occurs.Hepatic fibrosis mice is divided into two groups.First group is experimental group（21 mices）, will be injected
The monoclonal antibody of anti-human tGM2, the dosage of antibody is 0.005 milligram respectively, 0.02 milligram and 0.1 milligram, every kind of dosage injection
7 mices.Second group is matched group（7 mices）, normal rat IgG will be injected, dosage is 0.1 milligram.3rd group is
Blank group（7 mices）, mice is not through the injection of TAA and antibody.TAA injects latter 2 days the last time, through abdominal cavity note
Penetrate the monoclonal antibody of anti-human tGM2 or normal rat IgG.5 days afterwards, then injects a wheel antibody.After 5 days, mice quilt
Kill, therapeutic effect is evaluated.
Fig. 2A shows Picro-Sirius red（Sirius Red）The result of dyeing, is compared with matched group, and hepatic fibrosis knot exists
Anti-human tGM2 mab treatment group（0.1 milligram）Substantially reduce.Fig. 2 B shows, markers of fibrosis molecule alpha-smooth muscle flesh moves
Albumen（α-SMA）Expression reduce also with the increase of anti-human tGM2 antibody dosage.Fig. 2 C shows, in matched group, suppressed
The expression of MMP9 obtain part and recover, but in experimental group, the injection of anti-human tGM2 antibody is significantly enhanced the expression of MMP9
Level.This is consistent with disappearing of hepatic fibrosis in mice.
SEQUENCE LISTING
<110>Wuhan Xie Kang bio tech ltd
<120>A kind of monoclonal antibody of anti-human tissue T-5398 2 and its application
<130>A kind of monoclonal antibody of anti-human tissue T-5398 2 and its application
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 27
<212> PRT
<213> human
<400> 1
Gly Tyr Glu Gly Trp Gln Ala Leu Asp Pro Thr Pro Gln Glu Lys Ser
1 5 10 15
Glu Gly Thr Tyr Cys Cys Gly Pro Val Pro Val
20 25
Claims (3)
1. a kind of hybridoma it is characterised in that：Rat anti-human tTG 2（tGM2）Hybridoma XK-
1, CCTCC NO: C201312.
2. the antibody of the hybridoma secretion described in claim 1.
3. application in preparation treatment hepatic fibrosis in mice medicine for the antibody described in claim 2.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054416A (en) * | 2006-06-23 | 2007-10-17 | 陈志南 | HAb18GC2 monoclonal antibody and its light and heavy chain variable area genes, coding polypeptide and use |
| CN101629958A (en) * | 2008-07-17 | 2010-01-20 | 中国医学科学院肿瘤研究所 | Detecting method by TGM2, detecting kit and application thereof |
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| CN101054416A (en) * | 2006-06-23 | 2007-10-17 | 陈志南 | HAb18GC2 monoclonal antibody and its light and heavy chain variable area genes, coding polypeptide and use |
| CN101629958A (en) * | 2008-07-17 | 2010-01-20 | 中国医学科学院肿瘤研究所 | Detecting method by TGM2, detecting kit and application thereof |
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