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Anti prourokinase monoclonal antibody hybrid oma cell line and its application

A technology of hybridoma cell line and urokinase, which is applied in the direction of anti-enzyme immunoglobulin, enzyme, anti-animal/human immunoglobulin, etc., can solve the problems of complexity and low recovery rate, and achieve stable and specific antibody expression Strong sex, moderate affinity effect

Inactive Publication Date: 2006-09-13
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using this method to purify scu-PA, the preparation scale is relatively large, but it is more complicated, and the recovery rate is lower than that of antibody affinity chromatography, which is about 60% to 70%.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1. Establishment of mouse-mouse hybridoma cells expressing anti-single-chain prourokinase monoclonal antibody

[0016] 1. Immunization of mice

[0017] Use 0.5mL of 50 μg of prourokinase with a purity of 99% (measured by the S-2444 method, prepared by our research group, see Chinese patent ZL96119836.2, international patent main classification number C12N 15 / 58), and 0.5mL of Freund’s complete Adjuvant (Gibco, Cat No. 15721-012) was mixed well, intraperitoneally or subcutaneously multi-point injected Balb / c mice, 10 days later, with 0.5mL prourokinase (60μg) and Freund's incomplete adjuvant (Gibco Company, Cat No.15720-014) were fully mixed to immunize the mice again, and on the 20th day, the mice were directly immunized again with 0.8 mL of 100 μg of prourokinase. On the 28th day, the eyeballs were bled, and the antibody titer was measured. When the antibody titer reached at least 1:1000, the day before the fusion, the same dose was used to boost the immunizat...

Embodiment 2

[0032]Embodiment 2, application: simple and efficient method for purifying scu-PA

[0033] 1. Preparation of prourokinase monoclonal antibody

[0034] 1. Expansion of hybridoma cells

[0035] The hybridoma cell 3G7-anti-Pro-UK that can secrete the monoclonal antibody of anti-single-chain pro-urokinase was cultured with 100mL square bottle, and every bottle was added with 10mL medium, and the medium was DMEM / F12 (1:1), Add fetal bovine serum content of 5-10%. After the cells are full, transfer to 1000mL spinner bottle for culture. Generally, 3 square bottles can be transferred to one spinner bottle. Add 100mL 2% serum medium to the spinner bottle, and the rotation speed is 0.3rpm . After the cells are overgrown in the spinner bottle, the cells in the 3-5 bottle spinner bottle can be transferred to a 1.5L stirred flask for further culture, the medium volume is 300-500mL, and the Cytopore-2 porous microcarrier (Pharmacia Biotech AB, Sweden, CatNo.17-1271-02) to culture hybrido...

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Abstract

A monoclonal prourokinase antibody hybridoma cell line (CGMCC No.0999) is disclosed, which can be used to prepare the purified monoclonal prourokinase antibody. Its advantages are mederate affinity, high specificity, and stable genetic characteristics.

Description

technical field [0001] The present invention relates to an anti-prourokinase monoclonal antibody hybridoma cell line and its application, more precisely, it uses cell engineering and antibody engineering technology to obtain a mouse-mouse hybridoma cell line secreting an anti-urokinase monoclonal antibody, and through the hybridoma The porous microcarrier culture of cells, the purification of cell culture supernatant by affinity chromatography, and the preparation of monoclonal antibodies that can be used for purification of prourokinase and identification of its physical and chemical properties belong to the technical field of bioengineering. Background technique [0002] Thrombosis has seriously threatened human health. Prourokinase (Pro-UK) is also called single-chain urokinase-type plasminogen activator (single-chain urokinase-type plasminogen activator, scu-PA), and t- PA is also a second-generation thrombolytic drug with strong specificity, few side effects, and low re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C12N9/72C07K16/40C07K16/18
Inventor 高丽华肖成祖胡显文胥照平张正光王菲郭智霞
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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