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Single-domain antibody specific to coagulation factor IX (FIX)

A coagulation factor, a single technology, used in the field of medicine and biology

Active Publication Date: 2019-10-18
SUZHOU ALPHAMAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current analysis methods can only be used for post-translational modification and specific activity analysis of highly purified recombinant FIX in the late stage of development.

Method used

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  • Single-domain antibody specific to coagulation factor IX (FIX)
  • Single-domain antibody specific to coagulation factor IX (FIX)
  • Single-domain antibody specific to coagulation factor IX (FIX)

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0124] Preparation of Factor IX Depleted Plasma

[0125] In another aspect, the present invention provides a method for preparing a FIX-deficient blood sample, comprising

[0126] a) contacting a blood sample with a FIX-binding molecule of the invention, whereby FIX in said blood sample forms a complex with said FIX-binding molecule,

[0127] b) separating said complex from said blood sample, and

[0128] c) Harvest FIX-deficient blood samples.

[0129] In some embodiments, wherein the FIX-binding molecule is immobilized on a solid support. In some embodiments, the solid support is selected from the group consisting of: polyethylene, polystyrene, polypropylene, polysulfone, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, cellulose, cellulose Acetate, cross-linked dextran, cross-linked agarose, chitin, chitosan, cross-linked dextran, cross-linked alginic acid, silicones, fluoropolymers and other synthetic polymers , or magnetic media (such as magneti...

Embodiment 1

[0138] Example 1: Screening of anti-FIX heavy chain single domain antibody

[0139] 1.1. Library construction:

[0140] Lymphocytes were isolated from 14 camels that had not been immunized, and spleens from 5 camels and lymph nodes from 8 camels were removed. The RNA extraction kit provided by QIAGEN was used to extract total RNA from lymphocytes and tissues. Super- The Script III FIRST STRANDSUPERMIX kit was used to reverse-transcribe all the extracted RNA into cDNA according to the instructions, and use nested PCR to amplify the nucleic acid fragment encoding the variable region of the heavy chain antibody.

[0141] The nucleic acid fragment of the target heavy chain single domain antibody was recovered, and cloned into the vector pMECS for phage display using restriction endonucleases (purchased from NEB) PstI and NotI. The product was then electrotransformed into Escherichia coli electroporation competent cell TG1 to construct a non-immune single domain antibody phage dis...

Embodiment 2

[0147] Example 2: Preparation of anti-FIX single domain antibody-Fc fusion protein by expression in mammalian cells

[0148] 2.1. Preparation of vectors expressing FIX single domain antibody-Fc fusion protein

[0149] Design primers for PCR amplification of the FIX single domain antibody VHH fragment, and the DNA fragment encoding human IgG1-Fc (see figure 1 ) fusion, and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing FIX single domain antibody-Fc fusion protein in mammals. Among them, the amplification of different VHH fragments uses general primers, and the general primers are as follows:

[0150] Upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC (SEQ ID NO: 33)

[0151] Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG (SEQ ID NO: 34)

[0152] 2.2. Preparation of Fc fusion protein of FIX single domain antibody

[0153] The vector obtained in 2.1 was transfected into HEK293 cells for transient expression of the antibody. D...

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Abstract

The invention relates to the field of medical biology, discloses a single-domain antibody specific to a coagulation factor IX (FIX), particularly discloses a FIX binding molecule derived from the single-domain antibody, and especially discloses application in FIX detection and FIX-poor plasma preparation.

Description

technical field [0001] The invention relates to the field of medicine and biology, and discloses a single-domain antibody against blood coagulation factor IX (FIX). Specifically, the present invention discloses a FIX-binding molecule derived from the single-domain antibody and its use, especially for detecting FIX and preparing FIX-deficient plasma. Background technique [0002] Coagulation factors are various protein components involved in the blood clotting process. Its physiological function is to be activated when the blood vessel is bleeding, stick together with platelets and plug the leak on the blood vessel. This process is called coagulation. They are produced in part by the liver. Can be inhibited by coumarin. For unified naming, the World Health Organization numbers them with Roman numerals according to the order in which they were discovered, including coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, etc. [0003] Hereditary coagulation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/36C12N15/13C12N15/85G01N33/68G01N1/28
CPCC07K16/36C12N15/85G01N33/6854G01N1/28C07K2317/569C07K2317/565
Inventor 徐霆王玲恽丽红白玉汪皛皛
Owner SUZHOU ALPHAMAB
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