Hybridoma cell, monoclonal antibody generated by hybridoma cell and application of monoclonal antibody

A technology of hybridoma cells and monoclonal antibodies, applied in the field of monoclonal antibodies, to achieve the effect of inhibiting blood coagulation reaction and inhibiting thrombus formation

Inactive Publication Date: 2014-08-06
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently few studies on the prevention and treatment of members of the protein disulfide isomerase (PDI) family for the prevention and treatment of thrombotic diseases and disseminated intravascular coagulation

Method used

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  • Hybridoma cell, monoclonal antibody generated by hybridoma cell and application of monoclonal antibody
  • Hybridoma cell, monoclonal antibody generated by hybridoma cell and application of monoclonal antibody
  • Hybridoma cell, monoclonal antibody generated by hybridoma cell and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of monoclonal antibody hybridoma cells

[0042] 1. Material

[0043] Antigen: the amino acid sequence protein shown in SEQ ID NO:1, fully synthesized by the reagent company;

[0044] Test animals: BALB / C mice aged 8-10 weeks, about 17g;

[0045] Reagents: Freund's complete adjuvant and incomplete adjuvant (purchased from Promega);

[0046] Myeloma cells: SP2 / 0 cells (provided by the China Type Culture Collection);

[0047] Medium: HAT selective medium (purchased from Sigma);

[0048] 2. Method

[0049] Take 100 μg of the protein of the amino acid sequence shown in SEQ ID NO: 1 plus equal mass Freund’s complete adjuvant for the first subcutaneous injection of BALB / C mice, 21 days later, take 300 μg of the protein of the amino acid sequence of SEQ ID NO: 1 plus The second subcutaneous injection of the same quality Freund’s incomplete adjuvant was carried out to BALB / C mice. After 10 days, blood was collected from the tail and the ELISA indirect method was used ...

Embodiment 2

[0051] Example 2: Secreted production of DuIn antibody

[0052] After immunizing BALB / C mice with incomplete adjuvant for 1 to 2 weeks, they were injected 1×10 into the abdominal cavity of the mice. 6 A hybridoma cell with a deposit number of CGMCC NO. 9153, which produces ascites 7-10 days after inoculation with the hybridoma cell;

[0053] The ascites was extracted from the abdominal cavity of the BALB / C mice, placed in a 15ml centrifuge tube, and centrifuged at 2000 rpm for 10 min to precipitate blood cells and other impurities. The supernatant was transferred to a new centrifuge tube, stored in a -80°C refrigerator, and purified Before, use 0.1M PBS solution to dilute ascites at a ratio of 1:3;

[0054] Take 0.5ml Protein G into the adsorption column, add 20 times the column volume of 0.1M PBS buffer to equilibrate the column for sample loading;

[0055] Take the diluted ascites and add it to the purification column, each time 2-3ml, the ascites circulate through the column 3 time...

Embodiment 3

[0059] Example 3: DuIn antibody specific detection

[0060] Take ERp57fl / fl (control mice, the gene encoding the amino acid sequence protein shown in SEQ ID NO:1 is not knocked out) and PF4-Cre / ERp57fl / fl (platelet specific knock out the amino acid sequence shown in SEQ ID NO:1 Protein-encoding gene mouse) platelet lysate 200μg and ERp57 recombinant protein 50ng were subjected to polyacrylamide gel electrophoresis, then the protein was transferred to PVDF membrane and sealed with 5% skimmed milk solution at room temperature for 1 hour. Use TBS / T solution to dilute the anti-DuIn antibody to 1 μg / ml, put the blocked PVDF membrane into the antibody diluent, and incubate at room temperature for 2 hours. After the incubation, wash with TBS / T solution 3 times for 10 minutes each time, then add membrane to the fluorescently labeled secondary antibody dilution, and incubate at room temperature for 1 hour. After 1 hour, wash the membrane 3 times with TBS / T solution and observe with Odyss...

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Abstract

The invention relates to the field of biotechnology and discloses a hybridoma cell, a monoclonal antibody generated by the hybridoma cell and application of the monoclonal antibody. The collection number of the hybridoma cell is CGMCC (China General Microbiological Culture Collection Center) NO.9153. The hybridoma cell and the monoclonal antibody secreted by the hybridoma cell are prepared from a member of the optimized PDI (protein disulfide isomerase) protein family as antigen protein; the hybridoma cell can stably generate the monoclonal antibody resistant to protein with an amino acid sequence as shown in SEQ ID NO:1; the monoclonal antibody has a two-way inhibition function, can inhibit thrombogenesis and blood clotting reaction, and can be applied to preparation of drugs for treating thrombotic diseases and clotting diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hybridoma cell, a monoclonal antibody produced therefrom and applications thereof. Background technique [0002] Thrombus refers to a semi-clot-like substance formed on the surface of blood vessels or the intima of the heart during blood flow. A thrombus consists of insoluble fibrin, deposited platelets, accumulated white blood cells, and trapped red blood cells. Thrombosis is a multifactorial process involving many interacting genetic and environmental factors. Thrombotic diseases, such as coronary syndrome and deep vein thrombosis, are common clinically. Thrombotic disease belongs to cardiovascular and cerebrovascular diseases. The number of deaths due to cardiovascular and cerebrovascular diseases in the world reaches 12 million every year, which is equivalent to 1 / 4 of the total death toll in the world. It has become the number one killer of human life. [0003] Disseminated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40A61K39/395A61P7/02C12R1/91
Inventor 武艺周俊松陈凤梧
Owner SUZHOU UNIV
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