Humanized anti-TLR4 antibody Fab as well as preparation method and application thereof

An antibody, fully human technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem that antibodies have not been developed, have not been very effectively inhibited, and cannot effectively control the biological effects of endotoxin. And other issues

Inactive Publication Date: 2013-09-25
MILITARY MEDICINE INST OF NANJING MILITARY REGION
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Although there are related studies on TLR4, no antibody against human TLR4 has been developed, so the biological effects of endotoxin cannot be effectively controlled. Therefore, various inflammatory reactions mediated by TLR4 have not been effectively inhibited.

Method used

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  • Humanized anti-TLR4 antibody Fab as well as preparation method and application thereof
  • Humanized anti-TLR4 antibody Fab as well as preparation method and application thereof
  • Humanized anti-TLR4 antibody Fab as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0038] Construction of fully human Fab phage antibody library:

[0039] (1) Collect 50 samples of whole blood from patients with severe hepatitis, separate their lymphocytes, use Invitrogen Trizol to extract total RNA, use oligo(dT)20 as a primer, and reverse transcribe to generate cDNA.

[0040] (2) Use human antibody variable region amplification primers to perform PCR to amplify the heavy and light chain variable region genes respectively. The Fab gene was synthesized by Overlap PCR and connected with the expression vector pComb3XSS digested with SfiI endonuclease to construct the prokaryotic recombinant expression vector of Fab.

[0041] (3) Electrotransform competent Escherichia coli XL1-Blue, and finally constructed a capacity of 5.76×10 9 Fully human Fab antibody library.

[0042] Linking the constructed Fab gene with the pComb3xSS vector refers to performing double digestion with SfiI endonuclease on the purified and quantified Fab gene; after purification and quanti...

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Abstract

The invention discloses a humanized anti-TLR4 antibody Fab as well as a preparation method of the humanized anti-TLR4 antibody Fab and an application of the humanized anti-TLR4 antibody Fab in inhibition of TLR4-mediated inflammatory reaction and belongs to the field of biological pharmacy. The preparation method comprises the following steps of: separating B-lymphocytes from the whole blood of 50 patients with serious hepatitis, amplifying the heavy and light chain variable region genes of all antibodies, constructing a completely humanized Fab antibody library by adopting a genetic engineering method, carrying out enrichment screening on a phage antibody library by using a recombinant TLR4 protein so as to separate out anti-TLR4 specificity humanized Fab antibody, carrying out affinity purification on an expression product by a Protein L column, and then using the expression product for function analysis of antibodies. In-vitro test results show that the anti-TLR4 genetic engineering antibody Fab can obviously block the expression of LPS (Lipopolysaccharide)-induced human PBMC (Peripheral Blood Mononuclear Cell) cells on TNF-alpha, and the inhibition ratio reaches 76.25%.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a human phage antibody library, an anti-TLR4 genetically engineered antibody Fab and its heavy chain variable region gene, light chain variable region gene and its encoded protein, as well as the protein and its derivatives role in the inflammatory response. Background technique [0002] Septic shock is also called septic shock or toxic shock. It is a state of microcirculation disorder caused by pathogenic microorganisms and their toxins in the human body, leading to tissue hypoxia, metabolic disorders, cell damage and even multiple organ failure. It is characterized by severe disease, rapid progression and high mortality. The most important pathogenesis of septic shock is mainly related to a series of inflammatory factors caused by infected bacteria and their endotoxins. Lipopolysaccharide is the main component of bacterial endotoxin, a component of the cell wall of Gram-negat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P29/00
Inventor 朱进汪茂荣冯振卿
Owner MILITARY MEDICINE INST OF NANJING MILITARY REGION
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