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A method for preparing genetically engineered IgG antibody in Escherichia coli

A technology of Escherichia coli and genetic engineering, applied in the field of preparation of genetically engineered IgG antibodies, can solve the problems of regression phenomenon, loss of antibody secretion ability, instability of hybridoma cell lines, etc., and achieve strong innovation, broad market application value, library The effect of large capacity

Active Publication Date: 2018-02-06
厦门瑞百泰生物医药有限公司
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  • Abstract
  • Description
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Problems solved by technology

However, there are also many problems with monoclonal antibodies. The current monoclonal antibodies are all from mouse origin, so they will produce a strong HAMA response when they are treated and used in humans
This defect severely limits the application of monoclonal antibodies
The hybridoma cell lines screened by cell fusion are often unstable, easily affected by various factors and lose the ability to secrete antibodies, or the so-called reversion phenomenon occurs
In addition, monoclonal antibody production technology is very time-consuming and laborious
Especially when preparing certain specific antigens, mammals like mice are not easy to produce high-affinity antibodies, so that high-sensitivity detection of special antigen molecules cannot be achieved

Method used

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  • A method for preparing genetically engineered IgG antibody in Escherichia coli
  • A method for preparing genetically engineered IgG antibody in Escherichia coli
  • A method for preparing genetically engineered IgG antibody in Escherichia coli

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Experimental program
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Embodiment 1

[0031] Example 1 Preparation and identification of the natural phage antibody library of the present invention.

[0032] 1) Extraction of spleen total RNA

[0033] Spleen total RNA was extracted by Trizol method. The animal was sacrificed by pulling the neck, and the abdomen of the mouse was cut open with DEPC scissors, and the spleen was quickly taken out, placed in a mortar pre-cooled with liquid nitrogen, crushed and ground into powder by adding liquid nitrogen. Transfer the powder to a DEPC-treated EP tube, add 1 mL of Trizol and 200 µL of chloroform, shake vigorously to mix, centrifuge at 10,000 r / min for 5-10 min, extract the supernatant, add 2 times the volume of absolute ethanol or 0.6 times volume of isopropanol for RNA precipitation, after centrifugation, the precipitate was blown dry, and 50-100 µL of DEPC water was added to dissolve the RNA.

[0034] 2), RT-PCR and antibody gene amplification

[0035] Using the extracted spleen total RNA as a template, the first...

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Abstract

The invention belongs to the field of antibody engineering and particularly relates to a method for preparing genetic engineering IgG antibodies in escherichia coli. The method comprises the following steps of: by phage display panning and genetic engineering technologies, respectively constructing prokaryotic expression vectors of heavy chains and light chains of antibodies, then converting the constructed prokaryotic expression vectors into escherichia coli, and inducing the expression of genetic engineering IgG antibodies by IPTG (isopropyl-beta-d-thiogalactoside). The method has the advantages that the generated genetic engineering IgG antibodies can be applied to the immunoassay kits, so that immunoassay for residues such as food, agricultural product additives and pesticide / antibiotics and the like, pathogenic microorganisms and related medical fields is realized, and a foundation is laid for commercialized development and production of high-quality immunoassaykit.

Description

technical field [0001] The invention belongs to the field of antibody engineering, and in particular relates to a method for preparing genetically engineered IgG antibodies in Escherichia coli. Background technique [0002] Since the creation of monoclonal antibody technology in 1975, monoclonal antibodies with strong specificity can be secreted through cell hybridization and screening without restriction, and have been produced in large quantities. Due to the characteristics of strong specificity and high affinity of this antibody molecule, it is very popular among people. favorite. However, there are also many problems with monoclonal antibodies. The current monoclonal antibodies are all derived from mice, so they will produce a strong HAMA response when they are treated and used in humans. This defect severely limits the application of monoclonal antibodies. The hybridoma cell lines screened by cell fusion are often unstable, and are easily affected by various factors a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C07K16/00
CPCC07K16/00C12N15/70C12N2800/101
Inventor 郑曾杰王荣智陈志斌陈玲玲曾林茂顾小松
Owner 厦门瑞百泰生物医药有限公司
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