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Bi-functional monoclonal antibody capable of anti-human von willbrand factor-A3

A monoclonal antibody, functional technology, applied in the field of monoclonal antibodies

Inactive Publication Date: 2009-12-09
苏州苏大赛尔免疫生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the development and development of monoclonal antibodies that block the interaction between the A3 region of von Willebrand factor and collagen or the A1 region and platelet GPIb will not only block the formation of thrombus from the early stage of thrombosis, but may also be able to utilize This monoclonal antibody explores how the A3 region and A1 region regulate the hemostatic function through the change of its configuration, so as to further study the mechanism of arterial thrombosis and development, and open up a new field for the prevention and early treatment of thrombotic diseases; no other Report of unit-specific dual-function inhibitory anti-von Willebrand factor A3 domain monoclonal antibody

Method used

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  • Bi-functional monoclonal antibody capable of anti-human von willbrand factor-A3
  • Bi-functional monoclonal antibody capable of anti-human von willbrand factor-A3
  • Bi-functional monoclonal antibody capable of anti-human von willbrand factor-A3

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Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Preparation of Immunogen--Recombinant Human Von Willebrand Factor A3 Region (rVWF-A3)

[0041] (1) Design and synthesis of PCR primers: design and synthesize a pair of primers respectively located on the upstream and downstream sides of the cDNA sequence (594bp) encoding the VWF-A3 region (amino acid 1681-1877),

[0042] For the upstream primer, see the base sequence described in SEQ ID NO.1: 5'-AT AAG CTT TCC CCTGCA CCT GAC TGC-3', with a Hind III restriction site at the 5' end;

[0043] Downstream primer, see the base sequence described in SEQ ID NO.2: 5'-TG CTC GAG CCT AACAAA TCC AGA GCA-3', 5' end contains Xho I restriction site; The cDNA was used as a template for PCR amplification to obtain PCR products.

[0044] (2) Cloning and sequence analysis of the target gene: under the action of T4 DNA ligase, the purified PCR product was directly connected to the pUCm-T vector to obtain the pUCm-T-VWF-A3 recombinant plasmid. The resulting recombinant plasmid w...

Embodiment 2

[0048] Embodiment 2: the preparation of the monoclonal antibody of specificity anti-von Willebrand factor A3 region We apply conventional immunology method and hybridoma technique ( and Milstein, Nature, 215:495-497, 1975) to prepare the monoclonal antibodies of the present invention.

[0049] First, 8-week-old female Balb / c mice (Experimental Animal Center, Shanghai Academy of Sciences) were immunized with purified rVWF-A3 region protein for three times with four weeks between each time. The first two times were multi-point subcutaneous injection on the back plus intraperitoneal injection, and the third time was injection through the mouse tail vein plus intraperitoneal injection.

[0050] After the serum antibody titer of the immunized mice reached a high enough level, the animals were sacrificed and spleen cells were isolated. Using the standard monoclonal antibody cell fusion technology, the spleen cells of the immunized Balb / c mice were fused with the mouse SP2 / 0 myelom...

Embodiment 3

[0053] Example 3: Chemical properties of monoclonal antibodies of the present invention

[0054] (1) It was confirmed by double-diffusion immunoassay that the monoclonal antibodies SZ-123 and SZ-125 of the present invention both belong to IgG1 subclass.

[0055] (2) Inoculate monoclonal antibodies SZ-123 and SZ-125 in the peritoneal cavity of Balb / c mice to produce ascites, and use G-Sepharose-4B (Pharmacia company product) column to purify, and it is determined that every 1 mL of ascites can be purified to obtain IgG 3- 6mg.

[0056] (3) ELISA assay to determine the specificity of SZ-123 and SZ-125: recombinant VWF A1, A2, A3 (all expressed and purified in our laboratory) and pure human VWF (provided from Anhui Green Cross Biological Products Co., Ltd. FVIII concentrates, purified by Sepharose 4B gel chromatography) (2 μg / ml) were coated on 96-well plates. After overnight at 4°C and washed with PBS-0.05%-Tween 20, they were blocked with 2% BSA-PBS overnight. Purified monoc...

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Abstract

The invention relates to a hybridoma cell strain SZ-125(3D2) with the prevention number of CGMCC NO.2501 and a bi-functional monoclonal antibody capable of anti-human von willbrand factor-A3 secreted by the hybridoma cell strain. The monoclonal antibody is combined with the VWF A3 to block the combination of the VWF A3 and collagen and simultaneously influences the configuration of domain A1, thereby destroying the original domain A1 configuration capable of combining platelet GPIb alpha and achieving the aim of blocking the interaction of the VWF A1 and the GPIb alpha, so that not only combination of the VWF A3 and the collagen is blocked but also the combination of the VWF A1 and the platelet GPIb alpha is blocked. Thus, the monoclonal antibody can be used for preparing anti-artery thrombosis medicine with anti-platelet adhesion activity.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to a bifunctional monoclonal antibody against human von Willebrand factor A3 (VWFA3) region and a preparation method thereof. The present invention further relates to the use of the bifunctional antibody in preparing anti-platelet adhesion and antithrombotic drugs. Background technique [0002] With the aging of the population and the increase in bad eating habits and other factors, the incidence and mortality of thrombotic diseases such as myocardial infarction and ischemic stroke have ranked first. In-depth research on the mechanism of thrombosis and the development of new antithrombotic drugs are currently the urgent problems to be solved in the field of medicine. [0003] 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 abciximab (Reopro), a monoclonal an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18A61K39/395A61P7/02
Inventor 阮长耿赵益明
Owner 苏州苏大赛尔免疫生物技术有限公司
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