CN105732814A - Human-mouse chimeric monoclonal antibody for human von willebrand factor A3 region as well as preparation method and application of human-mouse chimeric monoclonal antibody - Google Patents
Human-mouse chimeric monoclonal antibody for human von willebrand factor A3 region as well as preparation method and application of human-mouse chimeric monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a human-mouse chimeric monoclonal antibody for a human von willebrand factor A3 region as well as a preparation method and application of the human-mouse chimeric monoclonal antibody. Specifically, the human-mouse chimeric monoclonal antibody comprises a mouse source heavy chain variable region, a mouse source light chain variable region, a human source heavy chain constant region and a human source light chain constant region, wherein the mouse source heavy chain variable region is as shown in SEQ ID NO:1; the mouse source light chain variable region is as shown in SEQ ID NO:2; the human source heavy chain constant region is selected from any one of heavy chain constant regions of IgG, IgM, IgA, IgE and IgD; the human source light chain constant region is selected from any one of light chain constant regions of kappa or lambda. The human-mouse chimeric monoclonal antibody can inhibit both combination of vWF and collagen and accumulation of thrombocyte, is good in antithrombus effect and is unlikely to have anti-mouse immunoreaction in human bodies, and moreover the probability of clinical bleeding risk can be greatly reduced.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of people's Mus chimeric mAb in anti-human von willebrand disease factor (vWF) A3 district with high specific, high affinity binding characteristic and high antithrombotic acitivity and its preparation method and application.
Background technology
Epidemiological study shows, in recent decades, along with the aging of the raising of material life and population, the harm of thrombotic disease highlights day by day.In certain pathological conditions, thrombosis can suppress or stop blood flowing completely, thus causing that cellularity is downright bad, and serious threat human health.
It is thrombotic necessary process that platelet plays key effect, platelet adhesion and gathering in the formation of thrombosis.There is platelet adhesion at atheromatous plaque place and gathering and thrombosis subsequently, play an important role in the pathogenic process of the disease such as thrombosis formation again of angina pectoris, acute myocardial infarction and postangioplasty.
In recent years, the development of molecular targeted agents has been promoted for platelet Study on Molecular Mechanism in thrombosis, wherein with the monoclonal antibody abciximab (abciximab) for platelet glycoprotein (GP) IIb/IIIa for representative, clinical practice is achieved with good antithrombotic therapy effect.But, owing to abciximab there is also the potential danger causing severe haemorrhage, particularly even more serious in asian population, therefore not yet in Asia popularization and application, hinder the clinical practice of this kind of medicine to a certain extent.
In order to overcome antithrombotic reagent to there is this defect of hemorrhage side effect, antiplatelet adheres to and rises as new antithrombotic therapy means.The adhesion of platelet and subendothelial collagen is the beginning step of platelet thrombosis, antiplatelet adhesive pharmaceutical carrys out the initial adhesion (namely stoping hematoblastic adhesion and activation at thrombotic commitment) of blocking platelet and collagen by suppressing collagen-vWF-platelet GPIb axle, can realize to antithrombus formation, be able to reducing again the purpose of hemorrhage side effect.Therefore, this kind of medicine has a good application prospect in the treatment to other angiemphraxises and thrombotic disease.
For mouse monoclonal antibody SZ-123 and the SZ-125 of collagen-vWF-platelet GPIb this novel targets of axle disclosed in Chinese invention patent CN101597595A, it can occur high specific, high affinity to be combined with people and macaque vWF ELISA A3 district, and then prevention platelet is in the adhesion of collagen surface and activation.But, owing to mouse monoclonal antibody may induce generation people's against murine immunoreation in human body, therefore this immunoreation not only can weaken or destroy therapeutic effect, in some instances it may even be possible to causing allergy or allergy in the patient, can significantly limit clinical practice.It addition, when treating thrombotic disease, it usually needs repetitively administered, thus more increase above-mentioned immunoreactive probability of happening.
Summary of the invention
For above-mentioned situation, the invention provides people's Mus chimeric mAb (MHC-SZ123) in a kind of anti-human von willebrand disease factor A3 district, it can not only block the formation of thrombosis from thrombotic commitment, and can reduce the reduction of drug effect and the probability of happening of hemorrhage side effect and the allergy caused by immunoreation or allergy.
Specifically, people's Mus chimeric mAb in the anti-human von willebrand disease factor A3 district of the present invention includes variable region of heavy chain, Mus source, Mus endogenous light chain variable region, people source CH, people's endogenous light chain constant region；Wherein:
The aminoacid sequence of variable region of heavy chain, described Mus source is such as shown in SEQIDNO:1；
The aminoacid sequence of described Mus endogenous light chain variable region is such as shown in SEQIDNO:2；
Described people source CH is selected from IgG(γ), IgM(μ), IgA(α), IgE(ε), IgD(δ) in any one CH；
Described people's endogenous light chain constant region is selected from kappa(κ) or lambda(λ) in any one constant region of light chain.
Owing in antibody molecule, the different subtype of heavy chain is closely related with its effector function, therefore it is it desired to chimeric monoclonal antibody and produces required effector function it is necessary to select corresponding CH.Preferably, in above-mentioned people's Mus chimeric mAb, described people source CH is selected from IgG1(γ 1), IgG3(γ 3), IgG4(γ 4) in any one CH；It is furthermore preferred that described people source CH is the CH of IgG1.
Preferably, in above-mentioned people's Mus chimeric mAb, described people's endogenous light chain constant region is kappa(κ) constant region of light chain.
On the other hand, the preparation method that the invention provides people's Mus chimeric mAb in above-mentioned anti-human von willebrand disease factor A3 district, it comprises the following steps:
(1) adopt cDNA end rapid amplifying (RACE, rapidamplificationofcDNAend) technology, utilize pair of primers HGSP1 and KGSP1, amplify the nucleotide sequence of heavy chain and variable region of light chain in coding Mus source functional antibody respectively；Wherein:
The nucleotide sequence of described primer HGSP1 and KGSP1 is respectively as shown in SEQIDNO:3 and SEQIDNO:4；
(2) engineered method is adopted, utilize two to primer p-VH-F and p-VH-R and p-VK-F and p-VK-R, connect corresponding with the nucleotide sequence of heavy chain in coding human antibody and constant region of light chain to heavy chain in the coding Mus source antibody obtained in step (1) and the nucleotide sequence of variable region of light chain respectively, after order-checking confirms, it is thus achieved that the heavy chain of chimeric monoclonal antibody and light chain expression vector；Wherein:
The nucleotide sequence of described primer p-VH-F, p-VH-R, p-VK-F, p-VK-R is respectively as shown in SEQIDNO:5, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8；
(3) the paramount efficient expression target of expression vector cotransfection obtained in step (2) is fitted together to the recipient cell of monoclonal antibody, through many time clonings and specificity screening, obtain the cell strain of the chimeric monoclonal antibody of high efficient expression target, collect cell culture supernatant, people's Mus chimeric mAb in purified acquisition anti-human von willebrand disease factor A3 district.
Preferably, in above-mentioned preparation method, the recipient cell of the chimeric monoclonal antibody of described high efficient expression target is rat bone marrow tumour cell or Chinese hamster ovary (CHO, Chinesehamsterovary) cell, it is preferable that Chinese hamster ovary celI.These cells are possible not only to synthesis, assembling and immunoglobulin,exocrine, also have the glycosylated function of immunoglobulin simultaneously.
Another further aspect, the invention provides the Chinese hamster ovary celI strain MHC-SZ123 of people's Mus chimeric mAb in a kind of high efficient expression anti-human von willebrand disease factor A3 district, it can suspension culture, and it being different from the adherent growth of common Chinese hamster ovary celI, this characteristic is significant for the expression improving chimeric monoclonal antibody.The preservation information of above-mentioned human mouse chimeric antibody Chinese hamster ovary celI strain MHC-SZ123 is as described below: depositary institution is China typical culture collection center (CCTCC), preservation address is China. Wuhan. and Wuhan University, the preservation time is on January 20th, 2016, and deposit number is CCTCCNO:C2015184.
Last aspect, the invention provides the application in preparing antithrombotic reagent of the people's Mus chimeric mAb in above-mentioned anti-human von willebrand disease factor A3 district.The chimeric monoclonal antibody (and corresponding fragment) of the present invention is possible not only to suppress in vitro platelet adhesion and thrombosis, it is also possible to suppress the circulation hemodynamic change caused due to blood platelet disorders adhesion and platelet thrombosis in laboratory animal body.This antibody can apply to the platelet thrombosis and the postangioplasty thrombosed situation again that prevent a variety of causes from causing.Such as: this antibody can be used for mammal or human body, pulmonary infarction, of short duration cerebral infarction can be prevented, dvt, after bypass operation of coronary artery, operation implant repair valve or blood vessel (autologous, xenogenic origin, or synthetic) when the formation of thrombosis；This antibody can be also used for prevention balloon dilatation, Atherosclerotic Vessels: Changes Observed during Coronary rotary-cut, laser or adopt other proper methods to carry out angioplasty in platelet aggregation and thrombosis.Administration time can in, art preoperative at angiopoiesis and postoperative.It is possible to prevent thrombosis after so processing, therefore decreases death that postangioplasty thrombosis causes, myocardial infarction accordingly and the generation of the complication such as PTCA or coronary artery bypass surgery treatment must be again carried out.
Preferably, in above-mentioned application, the active component in described antithrombotic reagent both can be the chimeric monoclonal antibody that is used alone, can be again chimeric monoclonal antibody and the combining of other thrombolytic drugs.Other thrombolytic drugs described include, but is not limited to plasminogen activator (such as tissue-type plasminogen activator, urokinase, streptokinase, rt-PA), anticoagulant or antiplatelet drug (such as aspirin, heparin or Coumarins anticoagulation).
Due to the utilization of technique scheme, the present invention compared with prior art has the advantage that
(1) owing to the immunoreactivity of antibody molecule is mainly produced by constant region, the chimeric monoclonal antibody therefore containing Ren Yuan constant region not easily produces the immunoreation of against murine in human body, improves the compliance of patient；
(2) people's Mus chimeric mAb of the present invention can carry out the combination of high specific, high affinity with people and macaque vWFA3 district, and utilizes this monoclonal antibody of FOLTS model validation of classics to have anti thrombotic action in macaque body；
(3) people's Mus chimeric mAb of the present invention can suppress the combination of vWF and collagen, suppressing again hematoblastic gathering, its antithrombotic biological activity is higher, when therefore may be used for PTCA and/or unstable angina pectoris, prevent the formation of acute thrombus, reduce the generation of cardiac ischemia event；
(4) novel targets that people's Mus chimeric mAb of the present invention is targeted is vWF, rather than platelet, therefore will not cause the prolongation in hematoblastic decline and bleeding time upon administration, greatly reduces the generation of clinical bleeding risk.
Accompanying drawing explanation
Fig. 1 is the electrophoretogram of 5 '-RACE products in embodiment one, and wherein L represents light chain result, and H represents heavy chain result, and M is DL2000DNAMarker.
Fig. 2 is chimeric monoclonal antibody expression plasmid collection of illustrative plates in embodiment one, and wherein heavy chain expression plasmid is expressed as pMH3-MHC-SZ123VH, and light chain expression plasmid is expressed as pMH3-MHC-SZ123VK.
Fig. 3 is chimeric monoclonal antibody expression plasmid enzyme action qualification figure in embodiment one.
Fig. 4 is that in embodiment two, shaking flask stream adds the SDS-PAGE expressing chimeric monoclonal antibody, and wherein A represents positive control (50 μ g/mL), and B represents culture fluid supernatant, and H represents heavy chain, and L represents light chain.
Fig. 5 is the SDS-PAGE identifying the chimeric monoclonal antibody of sterling in embodiment three, and wherein 1 represents firstling (concentration is 1g/L), and 2 represent second batch product (concentration is 2g/L), and 3 represent the 3rd batch of product (concentration is 1.5g/L).
Fig. 6 is the effect curve figure of vWF and the combination of collagen in chimeric monoclonal antibody vitro inhibition human plasma in embodiment four, and wherein SZ-123 represents mouse monoclonal antibody, and MHC-SZ123 represents that people Mus is fitted together to monoclonal antibody.
Fig. 7 is the effect curve figure of the platelet aggregation of chimeric monoclonal antibody vitro inhibition ristomycin induction in embodiment four, wherein a represents that MHC-SZ123(concentration is 8 μ g/mL), b represents that MHC-SZ123(concentration is 5 μ g/mL), c represents that MHC-SZ123(concentration is 3.5 μ g/mL), d represents negative control IgG.
Fig. 8 is that the chimeric monoclonal antibody of various dose in embodiment five and normal saline are to the macaque CFRs inhibitory action design sketch occurred.
Fig. 9 is the chimeric monoclonal antibody of various dose in the embodiment five inhibitory action design sketch to vWF in macaque body Yu the combination of collagen.
Figure 10 is that the chimeric monoclonal antibody of various dose in embodiment five is to the inhibitory action design sketch of the platelet aggregation of ristomycin induction in macaque body.
Figure 11 is that the chimeric monoclonal antibody of various dose in embodiment five is to the inhibitory action design sketch of platelet count in macaque body.
Figure 12 is that the chimeric monoclonal antibody of various dose in embodiment five is to the inhibitory action design sketch in bleeding time in macaque body.
Detailed description of the invention
Below with reference to drawings and the specific embodiments, the technical scheme in the present invention is made further description.Unless specifically indicated, the material that uses in the following example, reagent, instrument etc. all can be obtained by commercial means.
Embodiment one: the structure of people's Mus chimeric mAb eukaryon expression plasmid.
1, the structure of monoclonal antibody SZ-123 heavy chain and variable region of light chain cDNA:
The 5 '-RACE technology of employing, the variable region sequences of the heavy chain of cloning function antibody and light chain from the hybridoma cell strain SZ-123 of the Mus monoclonal antibody secreting anti-human vWFA3 district, wherein with the mRNA of the hybridoma cell strain of secreting Mus monoclonal antibody SZ-123 for masterplate, and designing reverse transcription specific primer HGSP1 and the KGSP1 of a set of heavy chain and light chain, its nucleotide sequence is respectively as shown in SEQIDNO:3 and SEQIDNO:4.The electrophoretogram of 5 '-RACE products is as it is shown in figure 1, be light chain (L) result on the right side of wherein, and left side is heavy chain (H) result.
Corresponding relation according to nucleotide sequence Yu amino acid coding, analyzes the reading frame of PCR primer, it is determined that the aminoacid sequence of corresponding variable region, the aminoacid sequence of its heavy chain and light chain is respectively as shown in SEQIDNO:1 and SEQIDNO:2.Feature according to immunoglobulin gene verifies that it is antibody sequence.
2, the structure of expression plasmid pMH3-MHC-SZ123VH and pMH3-MHC-SZ123VK:
With the sequence of 5 '-RACE sequencing result gained for template, design two is to primer (its nucleotide sequence is such as shown in SEQIDNO:5 to SEQIDNO:8), respectively with the PCR method variable region sequences by Mus source functional antibody gene and signal peptide sequence amplification, and will be connected in the expression vector pMH3 containing above-mentioned human antibody IgG1 and kappa constant region with same enzyme enzyme action after heavy chain variable region gene and chain variable region gene EcoRI and NotI enzyme action, obtain the heavy chain expression plasmid pMH3-MHC-SZ123VH and light chain expression plasmid pMH3-MHC-SZ123VK of chimeric monoclonal antibody.Through order-checking, expression plasmid identifies that sequence is correct, its plasmid map is as in figure 2 it is shown, enzyme action identifies figure as shown in Figure 3.
Embodiment two: the chimeric expression of monoclonal antibody, screening, suspension domestication and shaking flask stream add expression.
1, CHO-S cell electrotransfection method:
20 μ g recombiant plasmid (pMH3-MHC-SZ123VH, pMH3-MHC-SZ123VK) corotation enter 3 × 106Individual CHO-S cell, electricity turns reaction system: 200 μ L, CHO-S cell suspension+20 μ g plasmid+10 μ g salmon sperm dnas, and electricity turns reaction condition: 500V, 500us, 4 times.
2, the screening of overexpression cell line:
Electricity turns cell suspension be laid in the culture dish containing 10mL culture medium (DMEM/F12=1:1, containing 10%FBS).Adopt 2.4mg/LG418(Geneticin) cell carried out pressurization screening, select monoclonal to 96 orifice plates.Long to, when being paved with at the bottom of 80% hole, removing containing FBS culture fluid, add D/F culture fluid (expression culture fluid) with the dosage in 100 μ L/ holes until clone, detect expression after 48h.Diluted sample 10 times, ELISA detects a time cloning, selects high-expression clone to 24 orifice plates, diluted sample 30 times, and ELISA detects, and most high expressed amount is 5 μ g/mL.Unicellular paving two time cloning after being digested by high-expression clone, selects secondary high-expression clone by a colony screening method.Diluted sample 100 times, it is 23 μ g/mL that ELISA detects the most high expressed amount of orifice plate 48h.This clonal cell line (called after Chinese hamster ovary celI strain MHC-SZ123) carries out follow-up work as master cell bank.
3, suspend domestication:
Overexpression cell line is carried out the domestication that suspends, high expressed two time cloning Chinese hamster ovary celI strain MHC-SZ123 is transferred in T75 culture bottle and cultivates, when cell confluency degree to about 90%, by cell dissociation in T75, use B001 culture medium instead, after being placed in incubator quiescent culture 36h, collect cell and also count, by 1.0 × 106Individual/mL cell is transferred in 100mL shaking flask, adds 30mL suspending nutrient solution B001, prepares the domestication that suspends.Treating that cell is double and in good condition for every day, representing suspends tames successfully.
4, high expressing cell clone shaking flask stream adds expression:
The cell density of Chinese hamster ovary celI strain MHC-SZ123 is adjusted to 1.0 × 106Individual/mL, is inoculated in (containing 30mLB001) in 250mL triangular flask, carries out a batch experiment.The clone selecting most advantage of expression carries out 3L shaking flask stream and adds expression.Inoculum density is 2 × 106Individual/mL, observation of cell state, detected cell density, made cell density be always held at 2.0 × 10 every day6Individual/mL, until cumulative volume expands to 1L.Treat that cell density length is to 4.0 ~ 6.0 × 106Individual/mL time, cell is transferred to 34 DEG C of cultivations, starts stream and adds F001(supplementing culture medium), every day keep sugar concentration at about 3.0g/L.Stream adds expression 7 days, and centrifugal collection fermented liquid supernatant, expression is about 200mg/L.Shaking flask stream add express chimeric monoclonal antibody SDS-PAGE as shown in Figure 4.
5, the stability experiment of overexpression cell line:
Take the Chinese hamster ovary celI strain MHC-SZ123 adapting to suspension culture as experiment cell, and proceed as follows:
(1) cell number and Cell viability are calculated；
(2) adjusting cell concentration is 3 × 105Cell/mL, 15mL, joins in 100mL shaking flask, is placed on 37 DEG C of shaking tables and cultivates with the speed of 128rpm, within 3 days, be a generation altogether；
(3) after taking 3 days, the cell suspension in shaking flask carries out cell counting again, calculates the antibody concentration in the cell concentration in every milliliter of suspension, total cellular score, cell multiplication number, cell dead percentage ratio alive and every milliliter of suspension；
(4) adjust cell again to add in shaking flask for 2nd generation；By that analogy, cultivated for the 30th generation always.
Embodiment three: the purification of chimeric monoclonal antibody.
Adopt the chimeric monoclonal antibody of proteinA affinity chromatography purification, described in comprising the following steps that:
(1) proteinA affinity column is cleaned with the water of 3 ~ 5 times of column volumes；
(2) proteinA affinity column is balanced with 20mM phosphate buffer (PBS) (pH=7.0)；
(3) by the membrane filtration with 0.45 μm of the cell culture supernatant containing required purified monoclonal antibody, the proteinA affinity column by PBS balance is pumped into；
(4) with 20mMPBS(pH=7.0) wash post, until OD280<0.01；
(5)With 0.1M Glycine-HCl buffer (pH=3.0) eluting destination protein, collect eluting peak, and regulate the antibody-solutions collected with 1MTris-HCl buffer (pH=9.0), until pH value is neutral；
(6) with 10mMPBS(pH=7.2) dialysis desalting, obtain the chimeric monoclonal antibody of sterling.
The SDS-PAGE testing result of above-mentioned chimeric monoclonal antibody is as shown in Figure 5.The concentration of chimeric monoclonal antibody SZ-123, the A after diluted sample 20 times after adopting Bradford method to measure purification595It is 0.5327, according to standard curve (Y=0.0044X+0.1395, R2=0.9963) calculating, the concentration of destination protein is 1.01 ~ 2.04g/L.
Embodiment four: the extracorporeal biology functional examination of chimeric monoclonal antibody.
1, chimeric monoclonal antibody suppresses the combination of vWF and collagen in human plasma:
Gather 14 parts of human normal plasmas's (3.8% citrate anticoagulation) altogether to be used for carrying out external anticoagulant experiment.The Human plactnta type III collagen of acetic acid is coated on 96 orifice plates, then 100 μ L human normal plasmas (1:50 dilution) being simultaneously introduced with chimeric monoclonal antibody is coated in the hole of collagen, it is incubated 2h, the anti-human vWF TPPA of the rabbit of combining vWF horseradish peroxidase-labeled after washing in 37 DEG C.Experiment in vitro shows, chimeric monoclonal antibody can dose-dependently suppress the combination (as shown in Figure 6) of vWF and collagen in human plasma.
2, chimeric monoclonal antibody suppresses the platelet aggregation of ristomycin induction:
Gathering normal healthy people whole blood (3.8% sodium citrate anticoagulant), 100g is centrifuged 10min.Take PRP, by the chimeric monoclonal antibody of various concentration (8 μ g/mL, 5 μ g/mL, 3.5 μ g/mL) respectively with PRP incubation at room temperature 10min in advance, then respectively with ristomycin (1.25mg/mL) induced platelet aggregation.Result shows, chimeric monoclonal antibody can dose-dependently suppress the platelet aggregation (as shown in Figure 7) that ristomycin is induced.
Embodiment five: the internal antithrombus formation functional examination of chimeric monoclonal antibody.
Reopro antibody is mosaic type anti human platelet surface GPB/IIIa antibody, is mainly used in preventing when PTCA and/or unstable angina pectoris the formation of acute thrombus at present clinically, reduce cardiac ischemia event generation (N.Engl.J.Med.,1997(336): p1689-1697).For the internal anti thrombotic action of people's Mus chimeric mAb in clear and definite anti-human von willebrand disease factor A3 district, adopt and can well reflect that the Folts model of body thrombosis is tested.
1, laboratory animal:
Yunnan macaque (purchased from Suzhou Xishan Zhongke Experimental Animal Co., Ltd.), 20, male and female half and half, in 4 ~ 8 years old age, when experiment starts, body weight is 7.2 ~ 10.5kg.
2, experimental design:
With current clinical practice, with normal saline (N.S.) for negative control, proved the drug effect of chimeric monoclonal antibody and corresponding dose-effect relationship by the experiment of internal antithrombotic.
3, observation index:
Set up Folts model, observe 1 hour periodically blood flow before and after single intravenous injection administration and lower the change of (CFR) frequency, and observe administration Platelet counting (PLT), bleeding time (BT), plasma prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), the coagulation indexes such as Fibrinogen (Fg), in conjunction with the vWF level in blood plasma, the platelet aggregation quantitative result induced in conjunction with situation and ristomycin of vWF and collagen in blood plasma, the internal anti thrombotic action of the chimeric monoclonal antibody of integrated survey.Respectively 30min before administration, after administration 15,30,60,120min and gather blood sample in 24 hours, detect These parameters.All blood samples are with the sodium citrate anticoagulant of final concentration 0.38%.
4, experiment in vivo:
(1) in 20 macaque bodies, Folts model is established, experiment is divided into 4 groups, namely normal saline group (negative control), chimeric monoclonal antibody low (0.1mg/kg), in (0.3mg/kg), high (0.6mg/kg) three dosage groups, often group 5, route of administration is single iv bolus administration.
(2) for the inhibitory action of CFR: after being administered 1 hour, normal saline group, the chimeric basic, normal, high dosage group suppression ratio of monoclonal antibody respectively 0.0%, 29.4%, 58.0%, 73.1%, its result is as shown in Figure 8.Through one factor analysis of variance (ANOVA) it can be seen that the chimeric monoclonal antibody group of various dose is compared with normal saline group, for CFR suppression ratio difference highly significant (P< 0.01), and the inhibitory action of CFR presents obvious dosage correlation by chimeric monoclonal antibody.This experiments show that, the chimeric monoclonal antibody of the present invention can play significant anti thrombotic action in animal body, has the great potential developing into antithrombotic reagent.
(3) for the inhibitory action of vWF in blood plasma Yu the combination of collagen: within 15 minutes to 1 hour upon administration, the chimeric basic, normal, high dosage group of monoclonal antibody is held in peak value for the inhibitory action of vWF in blood plasma Yu the combination of collagen, and its suppression ratio is respectively close to 33%, 51% and 92%；Within 2 hours, its suppression ratio is kept at more than half (as shown in Figure 9) of peak.
(4) for the inhibitory action of platelet aggregation: within 30 minutes upon administration, the chimeric basic, normal, high dosage group of monoclonal antibody all reaches maximal percentage inhibition, respectively 25.8%, 43.4% and 62.1%, at this time point, be there is obvious dosage correlation (as shown in Figure 10) by chimeric monoclonal antibody in the inhibitory action of the platelet aggregation that ristomycin is induced.
(5) other index: about Testing index such as PLT, BT, PT, TT, APTT, Fg and vWF levels, each experimental group does not all observe the change (as shown in FIG. 11 and 12) with significant difference.
Claims (10)
1. people's Mus chimeric mAb in anti-human von willebrand disease factor A3 district, it includes variable region of heavy chain, Mus source, Mus endogenous light chain variable region, people source CH, people's endogenous light chain constant region；Wherein:
The aminoacid sequence of variable region of heavy chain, described Mus source is such as shown in SEQIDNO:1；
The aminoacid sequence of described Mus endogenous light chain variable region is such as shown in SEQIDNO:2；
Described people source CH is any one the CH in IgG, IgM, IgA, IgE, IgD；
Described people's endogenous light chain constant region is any one the constant region of light chain in kappa or lambda.
2. people's Mus chimeric mAb in anti-human von willebrand disease factor A3 district according to claim 1, it is characterised in that:
Described people source CH is any one the CH in IgG1, IgG3, IgG4.
3. people's Mus chimeric mAb in anti-human von willebrand disease factor A3 district according to claim 2, it is characterised in that:
Described people source CH is the CH of IgG1.
4. people's Mus chimeric mAb in anti-human von willebrand disease factor A3 district according to claim 1, it is characterised in that:
Described people's endogenous light chain constant region is the constant region of light chain of kappa.
5. a preparation method for people's Mus chimeric mAb in anti-human von willebrand disease factor A3 district according to claim 1, it comprises the following steps:
1) adopt cDNA end rapid amplifying technology, utilize pair of primers HGSP1 and KGSP1, amplify the nucleotide sequence of heavy chain and variable region of light chain in coding Mus source functional antibody respectively；Wherein:
The nucleotide sequence of described primer HGSP1 and KGSP1 is respectively as shown in SEQIDNO:3 and SEQIDNO:4；
2) engineered method is adopted, utilize two to primer p-VH-F and p-VH-R and p-VK-F and p-VK-R, connect corresponding with the nucleotide sequence of heavy chain in coding human antibody and constant region of light chain to heavy chain in the coding Mus source antibody obtained in step 1) and the nucleotide sequence of variable region of light chain respectively, after order-checking confirms, it is thus achieved that the heavy chain of chimeric monoclonal antibody and light chain expression vector；Wherein:
The nucleotide sequence of described primer p-VH-F, p-VH-R, p-VK-F, p-VK-R is respectively as shown in SEQIDNO:5, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8；
3) by step 2) in the recipient cell of the chimeric monoclonal antibody of the paramount efficient expression target of expression vector cotransfection that obtains, through many time clonings and specificity screening, obtain the cell strain of the chimeric monoclonal antibody of high efficient expression target, collect cell culture supernatant, people's Mus chimeric mAb in purified acquisition anti-human von willebrand disease factor A3 district.
6. preparation method according to claim 5, it is characterised in that:
The recipient cell of the chimeric monoclonal antibody of described high efficient expression target is rat bone marrow tumour cell or Chinese hamster ovary cell.
7. preparation method according to claim 6, it is characterised in that:
The recipient cell of the chimeric monoclonal antibody of described high efficient expression target is Chinese hamster ovary cell.
8. a Chinese hamster ovary cell strain for people's Mus chimeric mAb in high efficient expression anti-human von willebrand disease factor A3 according to claim 1 district, it is preserved in China typical culture collection center, and deposit number is CCTCCNO:C2015184.
9. the people's Mus chimeric mAb in anti-human von willebrand disease factor A3 district according to claim 1 application in preparing antithrombotic reagent.
10. application according to claim 9, it is characterised in that:
Active component in described antithrombotic reagent is people's Mus chimeric mAb in anti-human von willebrand disease factor A3 district according to claim 1 or its combining with other thrombolytic drugs；
Other thrombolytic drugs described include plasminogen activator, anticoagulant or antiplatelet drug.
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| CN116444674A (en) * | 2022-11-21 | 2023-07-18 | 苏州大学 | Anti-von Willebrand factor antibody and application thereof |
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