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A method for preparing interferon polymer conjugate IFN-poegma

A technology of high molecular polymers and conjugates, applied in the field of biomedicine, can solve the problems of reduced biological activity, low reaction yield, difficult control of binding sites and coupling stoichiometry, etc., to maintain biological activity and simple purification steps , Improve the effect of in vitro stability

Active Publication Date: 2020-07-14
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current PEGylated interferon has disadvantages such as low reaction yield, difficulty in controlling the binding site and conjugation stoichiometry, and severely reduced biological activity.

Method used

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  • A method for preparing interferon polymer conjugate IFN-poegma
  • A method for preparing interferon polymer conjugate IFN-poegma
  • A method for preparing interferon polymer conjugate IFN-poegma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Method for preparing interferon polymer conjugate IFN-POEGMA

[0064] 1. Preparation of interferon-initiator conjugate

[0065] 1. Preparation of IFN-LPETGGH 6 Fusion protein and transpeptidase Sortase A-H 6

[0066] 1) Preparation of recombinant bacteria

[0067] IFN-LPETGGH 6 The amino acid sequence of the fusion protein is sequence 2 in the sequence listing, and the nucleotide sequence of its coding gene is sequence 1 in the sequence listing;

[0068] Transpeptidase Sortase A-H 6 The amino acid sequence of is sequence 4 in the sequence listing, and the nucleotide sequence of its coding gene is sequence 3 in the sequence listing;

[0069] Express IFN-LPETGGH 6 The recombinant vector of the fusion protein is the IFN-LPETGGH shown in sequence 1. 6 The vector obtained by inserting the gene encoding the fusion protein into the Nde I and Eco RI restriction sites of pET-25b(+) vector was named pET-25b-IFN-LPETGGH6;

[0070] Express Sortase A-H 6 The recombinant vector is the...

Embodiment 2

[0132] Example 2. Functional verification of interferon polymer conjugate IFN-POEGMA

[0133] 1. In vitro biological activity and thermal stability test of IFN-POEGMA

[0134] In the present invention, the anti-cell proliferation activity of IFN-POEGMA (IFN-POEGMA2 prepared by the second method of Example 2 is selected, that is, the molecular weight is 66.2kDa for characterization, the same applies below) is measured by the MTT method. Human Burkitt’s B lymphoma cells (Daudi B) were chosen because they are highly sensitive to IFN-α2.

[0135] Daudi B cells were cultured in RMPI-1640 containing 15% FBS, 50U / mL penicillin and 50μg / mL streptomycin for a period of time, and then inoculated a certain concentration of cell suspension (50μL / well, 7500 cells in a 96-well plate). ), serially dilute samples of IFN-Br, Pegasys and purified IFN-POEGMA conjugate, add 50μL each to a 96-well culture plate, set up a negative control (without IFN-α2) and a blank control (with culture medium only) )...

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Abstract

Provided is a method for preparing an interferon-POEGMA conjugate, comprising the following step: polymerizing an interferon-initiator conjugate, OEGMA, CuCl, CuCl2, and 1,1,4,7,10,10-hexamethyltriethylenetetramine in a buffer solution to obtain an IFN-POEGMA conjugate.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for preparing an interferon polymer conjugate IFN-POEGMA. Background technique [0002] Proteins have been widely used in many fields such as biomedical imaging, targeted therapy and clinical diagnosis. The use of protein alone has problems such as short half-life and poor stability. The protein-polymer conjugate is prepared by linking the protein with the polymer, which can effectively improve the solubility, stability, pharmacokinetics and therapeutic efficacy of the protein and reduce its immunogenicity. The traditional method of synthesizing protein-polymer conjugates is to connect the pre-prepared polymer with the protein. There are often uncertain coupling sites, low efficiency, poor yield, difficult product separation, poor quality control, and difficult activity. Many problems such as maintaining. [0003] Interferon-α2 (IFN-α2) is a powerful inhibitor of virus ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/107C07K14/555C07K14/56C07K14/565C07K14/57C07K19/00A61K38/21A61K47/56A61P35/00
CPCA61K38/21C07K1/107C07K14/555C07K14/56C07K14/565C07K14/57C07K19/00
Inventor 高卫平胡瑾王贵林
Owner TSINGHUA UNIV