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

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

Human-mouse chimeric monoclonal antibody against human von Willebrand factor A3 region and its preparation method and application

technical field

The invention belongs to the field of biotechnology, in particular to a human-mouse chimeric monoclonal antibody against the A3 region of von Willebrand factor (vWF) with high specificity, high affinity binding properties and high antithrombotic activity and its Preparation methods and applications.

Background technique

Epidemiological studies have shown that in recent decades, with the improvement of material life and the aging of the population, the hazards of thrombotic diseases have become increasingly prominent. Under certain pathological conditions, thrombus can inhibit or completely stop blood flow, leading to cellular necrosis and seriously threatening human health.

Platelets play a key role in thrombus formation, and platelet adhesion and aggregation are necessary processes for thrombus formation. Platelet adhesion and aggregation and subsequent thrombus formation in atherosclerotic plaques play an important role in the pathogenesis of angina pectoris, acute myocardial infarction, and rethrombosis after angioplasty.

In recent years, research on the molecular mechanism of platelets in the process of thrombus formation has promoted the development of molecularly targeted drugs, among which the monoclonal antibody abciximab (abciximab) against platelet glycoprotein (GP) IIb/IIIa is the representative, clinically The application has achieved good antithrombotic treatment effect. However, because abciximab also has the potential risk of causing severe bleeding, especially in Asian populations, it has not been promoted and used in Asia, which hinders the clinical application of this type of drug to a certain extent.

In order to overcome the defect of bleeding side effects of antithrombotic drugs, antiplatelet adhesion is emerging as a new antithrombotic therapy. The adhesion of platelets to subendothelial collagen is the initial step of platelet thrombus formation, and antiplatelet adhesion drugs block the initial adhesion of platelets to collagen by inhibiting the collagen-vWF-platelet GPIb axis (that is, to prevent the early stage of thrombosis Adhesion and activation of platelets), can achieve the purpose of not only resisting thrombus formation, but also reducing bleeding side effects. Therefore, this class of drugs has a good application prospect in the treatment of other vascular obstruction and thromboembolic diseases.

Chinese invention patent CN101597595A discloses mouse-derived monoclonal antibodies SZ-123 and SZ-125 targeting the collagen-vWF-platelet GPIb axis as a new target, which can interact with human and macaque von Willebrand factor A3 regions Binding with specificity and high affinity, thereby preventing the adhesion and activation of platelets on the collagen surface. However, since the mouse-derived monoclonal antibody may induce human anti-mouse immune response in the human body, this immune response will not only weaken or destroy the therapeutic effect, but may even cause allergic or hypersensitivity reactions in patients, which greatly clinical application is limited. In addition, in the treatment of thromboembolic diseases, repeated administration is usually required, which further increases the occurrence probability of the above-mentioned immune reaction.

Contents of the invention

In view of the above situation, the present invention provides a human-mouse chimeric monoclonal antibody (MHC-SZ123) against human von Willebrand factor A3 region, which can not only block the formation of thrombus from the early stage of thrombus formation, but also It can reduce the reduction of drug efficacy and the occurrence probability of bleeding side effects and allergic reaction or hypersensitivity reaction caused by immune reaction.

Specifically, the human-mouse chimeric monoclonal antibody against human von Willebrand Factor A3 region of the present invention includes a mouse heavy chain variable region, a mouse light chain variable region, a human heavy chain constant region, a human source light chain constant region; wherein:

The amino acid sequence of the murine heavy chain variable region is shown in SEQ ID NO: 1;

The amino acid sequence of the murine light chain variable region is shown in SEQ ID NO: 2;

The human heavy chain constant region is a heavy chain constant region selected from any one of IgG (γ), IgM (μ), IgA (α), IgE (ε), and IgD (δ);

The human light chain constant region is any light chain constant region selected from kappa (κ) or lambda (λ).

Since the different subtypes of heavy chains in antibody molecules are closely related to their effector functions, if it is desired that chimeric mAbs produce the desired effector functions, the corresponding heavy chain constant regions must be selected. Preferably, in the above human-mouse chimeric monoclonal antibody, the human heavy chain constant region is a heavy chain constant region selected from any one of IgG1 (γ1), IgG3 (γ3), and IgG4 (γ4); More preferably, the human heavy chain constant region is IgG1 heavy chain constant region.

Preferably, in the above human-mouse chimeric monoclonal antibody, the human light chain constant region is a kappa (κ) light chain constant region.

In another aspect, the present invention provides a method for preparing the above-mentioned human-mouse chimeric monoclonal antibody against the A3 region of human von Willebrand factor, which comprises the following steps:

(1) Using rapid amplification of cDNA end (RACE, rapid amplification of cDNA end) technology, a pair of primers HGSP1 and KGSP1 were used to amplify the nucleotide sequences encoding the heavy chain and light chain variable regions of murine functional antibodies respectively; :

The nucleotide sequences of the primers HGSP1 and KGSP1 are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively;

(2) Using genetic engineering methods, using two pairs of primers p-VH-F and p-VH-R and p-VK-F and p-VK-R, respectively, the encoded mouse antibody obtained in step (1) The nucleotide sequences of the heavy chain and light chain variable regions of the medium and light chains are correspondingly connected with the nucleotide sequences encoding the heavy and light chain constant regions of the human antibody. After sequencing and confirmation, the heavy and light chains of chimeric monoclonal antibodies are obtained. chain expression vector; wherein:

The nucleotide sequences of the primers p-VH-F, p-VH-R, p-VK-F, p-VK-R are shown in SEQIDNO:5, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8 respectively ;

(3) Co-transfect the expression vector obtained in step (2) into recipient cells that highly express the target chimeric monoclonal antibody, and obtain a cell line that highly expresses the target chimeric monoclonal antibody after multiple cloning and specific screening, The supernatant of the cell culture solution was collected and purified to obtain a human-mouse chimeric monoclonal antibody against the region of human von Willebrand factor A3.

Preferably, in the above preparation method, the recipient cells highly expressing the target chimeric monoclonal antibody are mouse myeloma cells or Chinese hamster ovary (CHO, Chinese hamsterovary) cells, preferably CHO cells. These cells can not only synthesize, assemble and secrete immunoglobulins, but also have the function of glycosylation of immunoglobulins.

In yet another aspect, the present invention provides a CHO cell line MHC-SZ123 that highly expresses a human-mouse chimeric monoclonal antibody against human von Willebrand factor A3 region, which can be cultured in suspension, unlike ordinary CHO cells. Adherent growth, this feature is of great significance for increasing the expression of chimeric mAbs. The preservation information of the above-mentioned human-mouse chimeric antibody CHO cell line MHC-SZ123 is as follows: the preservation unit is the China Center for Type Culture Collection (CCTCC), the preservation address is China. Wuhan. Wuhan University, and the preservation time is January 20, 2016 date, the deposit number is CCTCCNO:C2015184.

In the last aspect, the present invention provides the application of the above human-mouse chimeric monoclonal antibody against human von Willebrand factor A3 region in the preparation of antithrombotic drugs. The chimeric monoclonal antibody (and corresponding fragments) of the present invention can not only inhibit platelet adhesion and thrombus formation in vitro, but also inhibit circulation blood flow changes caused by abnormal platelet adhesion and platelet thrombus formation in experimental animals. The antibody can be used to prevent platelet thrombosis caused by various reasons and re-thrombosis after angioplasty. For example: the antibody can be used in mammals or humans to prevent pulmonary embolism, transient ischemic stroke, deep vein thrombosis, coronary artery bypass surgery, surgical implantation of repair valves or blood vessels (autologous source, allogeneic source, or Synthetic); the antibody may also be used to prevent platelet aggregation and thrombosis during balloon dilatation, atherectomy, laser, or angioplasty performed by other appropriate methods . The timing of administration can be before, during and after angioplasty. This treatment can prevent thrombosis, thereby reducing the incidence of complications such as death caused by thrombosis after angioplasty, myocardial infarction, and the need to perform PTCA or coronary artery bypass surgery again.

Preferably, in the above application, the active ingredient in the antithrombotic drug can be a chimeric monoclonal antibody used alone, or a combination of chimeric monoclonal antibody and other thrombolytic drugs. The other thrombolytic drugs include (but are not limited to) plasminogen activators (such as tissue plasminogen activator, urokinase, streptokinase, recombinant tissue plasminogen activator), anticoagulant or anticoagulant Platelet drugs (such as aspirin, heparin, or coumarin-type anticoagulants).

Due to the application of the above-mentioned technical solution, the present invention has the following advantages compared with the prior art:

(1) Since the immunoreactivity of antibody molecules is mainly produced by the constant region, the chimeric mAb containing the human constant region is not easy to produce an anti-mouse immune response in the human body, which improves the patient's medication compliance;

(2) The human-mouse chimeric monoclonal antibody of the present invention can bind to human and macaque vWFA3 regions with high specificity and high affinity, and the classic FOLTS model has been used to confirm that the monoclonal antibody has antithrombotic effect in macaques;

(3) The human-mouse chimeric monoclonal antibody of the present invention can not only inhibit the binding of vWF to collagen, but also inhibit the aggregation of platelets, and has stronger antithrombotic biological activity, so it can be used for PTCA and/or unstable angina , to prevent the formation of acute thrombus and reduce the occurrence of cardiac ischemic events;

(4) The new target of the human-mouse chimeric monoclonal antibody of the present invention is vWF, not platelets, so after administration, it will not cause a decrease in platelets and prolong the bleeding time, greatly reducing the risk of clinical bleeding occur.

Description of drawings

Figure 1 is the electrophoresis image of the 5'-RACE product in Example 1, where L represents the result of the light chain, H represents the result of the heavy chain, and M represents the DL2000 DNA Marker.

Fig. 2 is a map of chimeric monoclonal antibody expression plasmids in Example 1, wherein the heavy chain expression plasmid is represented as pMH3-MHC-SZ123VH, and the light chain expression plasmid is represented as pMH3-MHC-SZ123VK.

Fig. 3 is a diagram of enzyme digestion identification of chimeric monoclonal antibody expression plasmid in Example 1.

Figure 4 is the SDS-PAGE electrophoresis image of the chimeric monoclonal antibody expressed in shake flasks in Example 2, where A represents the positive control (50 μg/mL), B represents the culture supernatant, H represents the heavy chain, and L represents the light chain .

Figure 5 is the SDS-PAGE electrophoresis image of the pure chimeric monoclonal antibody identified in Example 3, where 1 represents the first batch of products (concentration is 1g/L), 2 represents the second batch of products (concentration is 2g/L), 3 means the third batch of product (concentration is 1.5g/L).

6 is a graph showing the effect of chimeric monoclonal antibodies in vitro inhibiting the binding of vWF and collagen in human plasma in Example 4, wherein SZ-123 represents mouse-derived monoclonal antibodies, and MHC-SZ123 represents human-mouse chimeric monoclonal antibodies.

Figure 7 is a graph showing the effect of chimeric monoclonal antibodies in vitro inhibiting ristocetin-induced platelet aggregation in Example 4, where a represents MHC-SZ123 (concentration is 8 μg/mL), b represents MHC-SZ123 (concentration is 5 μg /mL), c indicates MHC-SZ123 (3.5 μg/mL concentration), d indicates negative control IgG.

Fig. 8 is a graph showing the inhibitory effect of different doses of chimeric monoclonal antibody and saline on the occurrence of CFRs in macaques in Example 5.

Fig. 9 is a graph showing the inhibitory effect of different doses of chimeric monoclonal antibodies on the binding of vWF and collagen in rhesus monkeys in Example 5.

Fig. 10 is a graph showing the inhibitory effect of different doses of chimeric monoclonal antibodies on ristocetin-induced platelet aggregation in rhesus monkeys in Example 5.

Fig. 11 is a graph showing the inhibitory effect of different doses of chimeric monoclonal antibodies on platelet count in rhesus monkeys in Example 5.

Fig. 12 is a graph showing the inhibitory effect of different doses of chimeric monoclonal antibodies on bleeding time in rhesus monkeys in Example 5.

detailed description

The technical solutions in the present invention will be further described below in conjunction with the drawings and specific embodiments. Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained through commercial means.

Example 1: Construction of human-mouse chimeric monoclonal antibody eukaryotic expression plasmid.

1. Construction of the heavy chain and light chain variable region cDNA of the monoclonal antibody SZ-123:

Using 5'-RACE technology, the variable region sequences of the heavy chain and light chain of the functional antibody were cloned from the hybridoma cell line SZ-123 secreting the mouse monoclonal antibody against the human vWFA3 region, in which the mouse monoclonal antibody SZ- The mRNA of 123 hybridoma cell lines was used as a template, and a set of heavy chain and light chain reverse transcription specific primers HGSP1 and KGSP1 were designed, the nucleotide sequences of which were shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The electropherogram of the 5’-RACE product is shown in Figure 1, in which the light chain (L) result is on the right and the heavy chain (H) result is on the left.

According to the corresponding relationship between the nucleotide sequence and the amino acid coding sequence, the reading frame of the PCR product was analyzed to determine the amino acid sequence of the corresponding variable region, and the amino acid sequences of its heavy chain and light chain were shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. According to the characteristics of the immunoglobulin gene, it was verified as an antibody sequence.

2. Construction of expression plasmids pMH3-MHC-SZ123VH and pMH3-MHC-SZ123VK:

Using the sequence obtained from the 5'-RACE sequencing results as a template, two pairs of primers (the nucleotide sequences of which are shown in SEQ ID NO: 5 to SEQ ID NO: 8) were designed, and the variable region of the murine functional antibody gene was respectively synthesized by PCR. The sequence and signal peptide sequence were amplified, and the heavy chain variable region gene and light chain variable region gene were digested with EcoRI and NotI and connected to the human antibody IgG1 and kappa chain constant region digested with the same enzymes. In the expression vector pMH3, the heavy chain expression plasmid pMH3-MHC-SZ123VH and the light chain expression plasmid pMH3-MHC-SZ123VK of the chimeric monoclonal antibody were obtained. The sequence of the expression plasmid was confirmed to be correct by sequencing.

Example 2: Expression, screening, suspension acclimatization and shake flask expression of chimeric monoclonal antibody.

1. CHO-S cell electrotransfection method:

20 μg of recombinant plasmids (pMH3-MHC-SZ123VH, pMH3-MHC-SZ123VK) were transferred into 3×10 ⁶ CHO-S cells, electroporation reaction system: 200 μL, CHO-S cell suspension + 20 μg plasmid + 10 μg salmon sperm DNA, Electroporation reaction conditions: 500V, 500us, 4 times.

2. Screening of high expression cell lines:

Spread the electroporated cell suspension in a Petri dish containing 10 mL of medium (DMEM/F12=1:1, containing 10% FBS). 2.4mg/LG418 (Geneticin) was used to screen the cells under pressure, and single clones were selected and transferred to a 96-well plate. When the clone grows to cover 80% of the bottom of the well, remove the FBS medium, add D/F medium (expression medium) at a dose of 100 μL/well, and detect the expression after 48 hours. Samples were diluted 10-fold, ELISA was used to detect one clone, and high-expression clones were selected and transferred to a 24-well plate, the sample was diluted 30-fold, and ELISA was used to detect the highest expression level of 5 μg/mL. After digesting the high-expression clones, the single cells were paved for secondary clones, and the secondary high-expression clones were selected according to the primary clone screening method. The sample was diluted 100 times, and the highest expression level in the well plate was 23 μg/mL after 48 hours in the ELISA test. The cloned cell line (named CHO cell line MHC-SZ123) was used as the original cell bank for follow-up work.

3. Suspension acclimation:

The high-expression cell line was suspended and domesticated, and the high-expression secondary clone CHO cell line MHC-SZ123 was transferred to a T75 culture flask for culture. When the cell confluence reached about 90%, the cells in T75 were digested and replaced with B001 medium. After standing in the incubator for 36 hours, the cells were collected and counted, and 1.0×10 ⁶ cells/mL were transferred to a 100 mL shake flask, and 30 mL of suspension culture medium B001 was added to prepare for suspension acclimation. When the cells doubled every day and were in good condition, it indicated that the suspension acclimatization was successful.

4. High expression cell clone shake flask fed-batch expression:

Adjust the cell density of CHO cell line MHC-SZ123 to 1.0×10 ⁶ cells/mL, inoculate them in 250 mL Erlenmeyer flasks (containing 30 mL B001), and conduct batch experiments. The clone with the most expression advantage was selected for fed-batch expression in a 3L shake flask. The inoculation density was 2×10 ⁶ cells/mL, and the cell state was observed every day to detect the cell density, so that the cell density was kept at 2.0×10 ⁶ cells/mL until the total volume expanded to 1 L. When the cell density reaches 4.0~6.0×10 ⁶ cells/mL, transfer the cells to 34°C for culture, start feeding F001 (supplementary medium), and keep the sugar concentration at about 3.0g/L every day. Feed-batch expression was performed for 7 days, and the supernatant of the fermentation broth was collected by centrifugation, and the expression level was about 200 mg/L. Figure 4 shows the SDS-PAGE electrophoresis of the chimeric monoclonal antibody expressed in the shake flask.

5. Stability experiment of high expression cell lines:

Take the CHO cell line MHC-SZ123 adapted to suspension culture as the experimental cells, and perform the following operations:

(1) Calculate the number of cells and cell viability;

(2) Adjust the cell concentration to 3×10 ⁵ cells/mL, 15 mL in total, add to a 100 mL shaker flask, place on a shaker at 37°C at a speed of 128 rpm, and culture for 3 days as one generation;

(3) Take the cell suspension in the shake flask after 3 days and count the cells, and calculate the cell concentration, total number of cells, cell doubling number, percentage of dead cells and antibody concentration per ml of suspension;

(4) Then adjust the cells and add them to the shake flask as the second passage; and so on, cultivate until the 30th passage.

Example 3: Purification of chimeric monoclonal antibody.

The chimeric monoclonal antibody was purified by protein A affinity chromatography, and the specific steps were as follows:

(1) Wash the protein A affinity chromatography column with 3 to 5 times the column volume of water;

(2) Equilibrate the protein A affinity chromatography column with 20mM phosphate buffer (PBS) (pH=7.0);

(3) Filter the cell culture supernatant containing the desired purified monoclonal antibody with a 0.45 μm filter membrane, and pump it into the protein A affinity column that has been equilibrated with PBS;

(4) Wash the column with 20mMPBS (pH=7.0) until OD ₂₈₀ <0.01;

(5 ) Elute the target protein with 0.1M glycine-HCl buffer (pH=3.0), collect the elution peak, and adjust the collected antibody solution with 1M Tris-HCl buffer (pH=9.0) until the pH value is neutral;

(6) Dialyze and desalt with 10mMPBS (pH=7.2) to obtain pure chimeric monoclonal antibody.

The SDS-PAGE detection results of the above chimeric monoclonal antibodies are shown in FIG. 5 . The concentration of the purified chimeric monoclonal antibody SZ-123 was determined by the Bradford method. The A ₅₉₅ of the sample diluted 20 times was 0.5327. According to the standard curve (Y=0.0044X+0.1395, R ² =0.9963), the concentration of the target protein was 1.01~2.04g/L.

Example 4: In vitro biological function determination of chimeric monoclonal antibody.

1. Chimeric monoclonal antibody inhibits the binding of vWF and collagen in human plasma:

A total of 14 normal human plasma (anticoagulated with 3.8% citrate) were collected for in vitro anticoagulation experiments. Coat acetic acid-acidified human placental type III collagen on a 96-well plate, then add 100 μL of normal human plasma (diluted at 1:50) and chimeric monoclonal antibody to the collagen-coated wells at the same time, and incubate at 37°C for 2 hours. After washing, bound vWF was detected with horseradish peroxidase-labeled rabbit anti-human vWF antibody. In vitro experiments showed that the chimeric mAb could dose-dependently inhibit the binding of vWF to collagen in human plasma (as shown in Figure 6).

2. Chimeric monoclonal antibody inhibits platelet aggregation induced by ristocetin:

Whole blood (anticoagulated with 3.8% sodium citrate) was collected from normal healthy people, and centrifuged at 100g for 10min. Take PRP, incubate chimeric monoclonal antibodies of various concentrations (8 μg/mL, 5 μg/mL, 3.5 μg/mL) with PRP at room temperature for 10 minutes, and then induce platelets with ristocetin (1.25 mg/mL). gather. The results showed that the chimeric mAb could dose-dependently inhibit ristocetin-induced platelet aggregation (as shown in Figure 7).

Example 5: Determination of antithrombotic function of chimeric monoclonal antibody in vivo.

Reopro antibody is a chimeric anti-GP on the surface of human platelets b/IIIa antibody is currently clinically used to prevent acute thrombosis and reduce the occurrence of cardiac ischemic events in PTCA and/or unstable angina pectoris ( N.Engl.J.Med . , 1997 (336):p1689 -1697). In order to clarify the in vivo antithrombotic effect of the human-mouse chimeric monoclonal antibody against the A3 region of human von Willebrand factor, the Folts model, which can well reflect the process of thrombus formation in the body, was used to conduct experiments.

1. Experimental animals:

Yunnan rhesus monkeys (purchased from Suzhou Xishan Zhongke Experimental Animal Co., Ltd.), 20 males and females, aged 4-8 years, weighed 7.2-10.5 kg at the beginning of the experiment.

2. Experimental design:

With the current clinical application, normal saline (N.S.) is used as a negative control, and the antithrombotic experiment in vivo is used to prove the efficacy of the chimeric monoclonal antibody and the corresponding dose-effect relationship.

3. Observation indicators:

Establish the Folts model, observe the changes in the frequency of cyclical flow reduction (CFR) within 1 hour before and after a single intravenous injection, and observe the platelet count (PLT), bleeding time (BT) and plasma prothrombin time before and after administration (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fg) and other coagulation indicators, combined with the level of vWF in plasma, the combination of vWF and collagen in plasma, and the Quantitative results of platelet aggregation induced by mycomycin were used to comprehensively investigate the antithrombotic effect of chimeric monoclonal antibody in vivo. Blood samples were collected 30 minutes before administration, 15, 30, 60, 120 minutes and 24 hours after administration to detect the above indicators. All blood samples were anticoagulated with sodium citrate at a final concentration of 0.38%.

4. In vivo experiment:

(1) The Folts model was established in 20 rhesus monkeys. The experiment was divided into 4 groups, namely, normal saline group (negative control), chimeric monoclonal antibody low (0.1mg/kg), medium (0.3mg/kg), high ( 0.6mg/kg) in three dose groups, 5 rats in each group, and the route of administration was a single intravenous injection.

(2) Inhibitory effect on CFR: 1 hour after administration, the inhibition rates of the normal saline group, the low-, medium-, and high-dose chimeric mAb groups were 0.0%, 29.4%, 58.0%, and 73.1%, respectively. The results are shown in the figure 8. According to the one-way analysis of variance (ANOVA), the difference in the inhibition rate of CFR between different doses of chimeric monoclonal antibody group and normal saline group was very significant ( P <0.01), and the inhibitory effect of chimeric monoclonal antibody on CFR showed There was a clear dose dependence. This experiment shows that the chimeric monoclonal antibody of the present invention can exert a significant antithrombotic effect in animals, and has great potential to be developed into an antithrombotic drug.

(3) Inhibition of the combination of vWF and collagen in plasma: within 15 minutes to 1 hour after administration, the low, medium and high dose groups of chimeric monoclonal antibody had no inhibitory effect on the combination of vWF and collagen in plasma. Maintained at the peak, the inhibition rates were close to 33%, 51% and 92%; within 2 hours, the inhibition rates were maintained at more than half of the maximum value (as shown in Figure 9).

(4) Inhibitory effect on platelet aggregation: Within 30 minutes after administration, the chimeric mAb low, medium and high dose groups all reached the maximum inhibition rate, which were 25.8%, 43.4% and 62.1%, respectively. Point, there is a clear dose-dependent effect of chimeric monoclonal antibody on ristocetin-induced platelet aggregation (as shown in Figure 10).

(5) Other indicators: Regarding the detection indicators such as PLT, BT, PT, TT, APTT, Fg and vWF levels, no statistically significant changes were observed in each experimental group (as shown in Figures 11 and 12).

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.