Variable region gene of full human monoclonal antibody specific to pro-protein convertase subtilisin/kexin 9 (PCSK9) and application thereof

A monoclonal antibody, fully human technology, applied in applications, antibodies, genetic engineering, etc., can solve problems such as abnormal liver enzymes, rhabdomyolysis, statin intolerance, etc.

Active Publication Date: 2015-08-26
ZHEJIANG BLUE SHIELD PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, statins have side effects such as abnormal liver enzymes and rhab

Method used

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  • Variable region gene of full human monoclonal antibody specific to pro-protein convertase subtilisin/kexin 9 (PCSK9) and application thereof
  • Variable region gene of full human monoclonal antibody specific to pro-protein convertase subtilisin/kexin 9 (PCSK9) and application thereof
  • Variable region gene of full human monoclonal antibody specific to pro-protein convertase subtilisin/kexin 9 (PCSK9) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of Human Monoclonal Antibody Phage Display Library

[0037] The present invention first constructs a natural human Fab phage display library according to the report of Hans J.W.de Haard et al. 10 , the host is Escherichia coli TG1.

Embodiment 2

[0038] Example 2, screening the Fab phage library to obtain a positive monoclonal anti-human PCSK9

[0039] 1. Dilute PCSK9 protein (purchased from GenScript Biotechnology Co., Ltd.) with coating buffer (PBS) to 100 μg / ml; take 2 ml into the immunotube, and coat overnight at 4°C; the next day, discard the supernatant, Wash the tube 3 times with PBS; 2% MPBS (PBS containing 2% skimmed milk powder), block at room temperature for 2h; discard the blocking solution; phage antibody library (10 12 ~10 13 pfu) was suspended in 2ml 2% MPBS, placed on a turntable and repeatedly turned over, and sealed at room temperature for 1 hour; the blocked phage antibody library was added to a PCSK9-coated immune tube, placed on a turntable and turned over repeatedly for 30 minutes, and then allowed to stand at room temperature 90min; wash the tube 20 times with PBS containing 0.1% Tween-20, then wash the tube 20 times with PBS; add 1ml of 100mM triethylamine, place on a turntable and incubate for...

Embodiment 3

[0043] Example 3, Expression and Purification of Positive Monoclonal PA4 Antibody Fab Fragment

[0044] 1. Select the PA4-infected TG1 strain for culture, use the plasmid mini-extraction kit to extract, and collect the PA4 phagemid vector. According to the report of Schoonbroodt S et al. (Nucleic Acids Research, Schoonbroodt S, 2005, 33, e81), the phagemid vector was digested with MluI restriction endonuclease to remove Gene III.

[0045] 2. Transform the obtained GeneIII excised plasmid into Escherichia coli TG1, 2YTA medium, culture to logarithmic phase at 37°C, add 1mM IPTG, induce expression overnight at 30°C.

[0046] 3. Collect the supernatant by centrifugation, and use Protein A to purify to obtain PA4Fab protein.

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PUM

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Abstract

The invention relates to the technical field of genetically engineered antibodies, in particular to a full human monoclonal antibody specific to pro-protein convertase subtilisin/kexin 9 (PCSK9) and a preparation method and an application of a full length antibody. According to a variable region gene, at least 80% identity between variable region code polypeptide of a heavy chain of the gene and a sequence that is shown in SEQ ID NO.1 can be achieved, and at least 80% identity between variable region code polypeptide of a light chain of the gene and a sequence that is shown in SEQ ID NO.2 can be achieved. Specifically, a human monoclonal antibody phage display library is used for screening Fab antibody specific to human PCSK9, and a complete full length antibody is obtained by a genetic engineering method. The antibody can be specifically combined with PCSK9, interaction of PCSK9 and low density lipoprotein receptor (LDLR) is effectively affected, LDLR degradation is inhibited, and the antibody can be applied to curing cholesterol and lipid metabolism diseases.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a new high-affinity fully human Fab antibody and full-length antibody that can specifically bind to PCSK9, which can weaken or block the interaction between PCSK9 and low-density lipoprotein receptor (LDLR) Interacting, inhibiting PCSK9-mediated LDLR degradation, thereby assisting in increasing the uptake of low-density lipoprotein cholesterol (LDL-C), is a genetically engineered antibody with hypolipidemic activity. Background technique [0002] Hypercholesterolemia is an important pathogenic factor of cardiovascular disease. A large number of clinical studies have shown that reducing low-density lipoprotein cholesterol (LDL-C) levels can help reduce the risk of cardiovascular disease and improve clinical outcomes. Statins are the most widely used lipid-lowering drugs in clinical practice. As inhibitors of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, statins c...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13A61K39/395A61P3/06
Inventor 郭志刚曹艳高婷婷杨欢
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD
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