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Human-derived anti-human tissue factor Fab and preparation method thereof

A technology of tissue factor and human antibody, which is applied in the field of therapeutic drugs for atherosclerosis, anti-human tissue factor Fab fragment, and Fab antibody, which can solve the problems of bleeding, lack of anticoagulant effect, etc., and reduce immunogenicity , the effect of weakening the effectiveness

Inactive Publication Date: 2009-05-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Many anticoagulant drugs that have been used so far, including heparin drugs, platelet aggregation inhibitors, thrombin inhibitors, etc., all target proteins located in the middle and lower reaches of the blood coagulation pathway, and theoretically they cannot achieve the most ideal anticoagulant effect. Coagulation, and adverse reactions such as bleeding caused by excessive inhibition may occur during use

Method used

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  • Human-derived anti-human tissue factor Fab and preparation method thereof
  • Human-derived anti-human tissue factor Fab and preparation method thereof
  • Human-derived anti-human tissue factor Fab and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Construction of Antibody Library

[0055] Select 300 informed healthy people who have not suffered from infectious diseases such as colds and chronic diseases in the past 2 months, take 5ml of peripheral anticoagulant blood each, separate lymphocytes with Ficoll-Paque, mix them for the construction of antibody library, and extract the total RNA. Use the Gene-Amp RNA PCR Kit to total RNA with Oligo(dT) 16 Reverse transcribe into cDNA, and carry out PCR amplification of immunoglobulin γ, κ, λ chain genes with the upstream and downstream primers (Table 1) of the conserved sequence of the human IgG light and heavy chain variable region.

[0056] After the PCR products were purified, the κ chain and λ chain products were double-digested with Asc I and Nhe I, respectively. The digested κ chain and λ chain products were connected to the human immunoglobulin Fab expression vector pFab-His2 ( figure 1 , figure 2 ), and then electroporated into JM109 competent cel...

Embodiment 2

[0062] Example 2: Antibody Library Screening

[0063] (1) ELISA identification:

[0064] Transform 100ul JM109 competent cells with 1ul plasmid from the constructed antibody library, spread evenly on LB plates containing 100ug / ml ampicillin, and culture at 37°C for 12-16h, until monoclonal colonies grow. Pick out the monoclonal colony and the negative control (transformation of JM109 competent cells with the pFab1-His2 plasmid without heavy and light chain inserted) respectively from the LBA plate, and inoculate them in 2ml of SBA (30 g of tryptone, 20 g of yeastextract, 10 g of MOPS per liter, 100ug / ml Ampicillin, pH7.0) culture medium, the backup plate was cultured at 37°C overnight, and stored at 4°C. Bacteria cultured to OD 600 =0.5~0.8, add IPTG with a final concentration of 0.1mM and induce overnight at 30°C. The next morning, centrifuge at 8000rpm×15min, discard the supernatant, resuspend in 250ulPBS (containing PMSF with a final concentration of 1mM), and centrifuge...

Embodiment 3

[0083] Example 3 hTFFab 148 Expression, purification and identification of

[0084] (1) hTFFab 148 expression and purification of

[0085] hTFFab 148 After the cloning plasmid is transformed into JM109 competent cells, spread it evenly on the LBA plate. After culturing overnight, pick a single clone colony and inoculate it in 10ml of SBA culture medium with shaking at 37°C×250rpm until OD 600 About 1.0, dilute to 1000ml SBA culture medium at 37℃×250rpm according to the ratio of 1:100 and continue to shake to OD 600 About 1.0, inoculated in 18L SBA medium in a 30L fermenter, 37℃×350rpm, pH7.0, dissolved oxygen 60%, continue to culture to OD 600 Add IPTG to a final concentration of 0.5mM at about 1.0 and induce expression at 30°C for 8h ( Figure 8 ), 4000rpm×30min, centrifuge at 4°C, collect the bacteria, and freeze them at -80°C.

[0086] According to the wet weight of bacteria, add 5ml NPI-10 (containing PMSF with a final concentration of 1mM) per gram of bacteria to re...

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Abstract

The invention belongs to the biological technical field, and relates to an anti-human tissue factor Fab fragment with the function of anticoagulation screened by using natural humanized antibody gene bank. The invention screens out the humanized anti-human tissue factor Fab, namely hTFFab148 from a bank by establishing a humanized antibody gene bank and ELISA, diluting thrombin time, and order-checking analysis, and the like. A large amount of expression purification and further authentication prove that the humanized anti-tissue factor Fab antibody has anticoagulating activity, does not generate unwanted side effect which is related to the prior anticoagulating combined drug, and has the property superior to the TF antibody of the prior art.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to the application of natural human antibody gene library to screen anti-human tissue factor Fab fragments with anti-coagulant function. The present invention relates to the DNA encoding the above-mentioned Fab antibody, especially the V region fragment of the antibody, and the DNA encoding the L chain or H chain containing the V region. The present invention also relates to a recombinant vector containing the DNA and a host transformed with the vector. In addition, the present invention also relates to a pharmaceutical composition and a therapeutic drug for atherosclerosis with the human antibody against human TF as an active ingredient. Background technique [0002] Tissue factor (TF), also known as coagulation factor III, is a transmembrane glycoprotein with a molecular weight of 47KD. The TF / FVIIa complex starts the extrinsic blood coagulation pathway; it can also activate c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/36C12N15/63C12N1/21C12N5/10C12N1/19C12R1/19C12R1/645
Inventor 马端程训佳刘静王羽雄木金贵梁旺王际平
Owner FUDAN UNIV
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