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Preparation and application of interferon polymer conjugate ifn-pmpc

A technology of polymer conjugates and polymer monomers, 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 purify The steps are simple and the effect of improving the stability in vitro

Active Publication Date: 2020-12-29
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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  • Preparation and application of interferon polymer conjugate ifn-pmpc
  • Preparation and application of interferon polymer conjugate ifn-pmpc
  • Preparation and application of interferon polymer conjugate ifn-pmpc

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, the method for preparing interferon polymer conjugate body IFN-PMPC

[0079] 1. Preparation of interferon-initiator conjugates

[0080] 1. Preparation of IFN-LPETGGH 6 Fusion protein and transpeptidase Sortase A-H 6

[0081] 1) Preparation of recombinant bacteria

[0082] IFN-LPETGGH 6 The amino acid sequence of the fusion protein is sequence 2 in the sequence listing, and the nucleotide sequence of the gene encoding it is sequence 1 in the sequence listing;

[0083] Transpeptidase Sortase A-H 6 The amino acid sequence is sequence 4 in the sequence listing, and the nucleotide sequence of the gene encoding it is sequence 3 in the sequence listing;

[0084] 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 the pET-25b(+) vector is named pET-25b-IFN-LPETGGH 6 ;

[0085] Express...

Embodiment 2

[0152] Example 2, Functional verification of the interferon polymer conjugate IFN-PMPC

[0153] 1. In vitro biological activity of IFN-PMPC

[0154] In the present invention, the anti-cell proliferation activity of IFN-PMPC (select the IFN-PMPC prepared by the second method of Example 2, that is, the molecular weight is 57kDa, the same below) is determined by the MTT method. Human Burkitt's B lymphoma cells (Daudi B) were chosen because they are highly sensitive to IFN-α2.

[0155] After Daudi B cells were cultured in RMPI-1640 containing 15% FBS, 50 U / mL penicillin and 50 μg / mL streptomycin for a period of time, a certain concentration of cell suspension (50 μL / well, 7500 cells) was inoculated in a 96-well plate ), the IFN-LPETGGH 6 , IFN-Br, IFN-α, Pegasys and the purified IFN-PMPC conjugate samples were serially diluted, each 50 μL was added to a 96-well culture plate, and a negative control (without IFN-α2) and a blank control (only containing medium), 37°C, 5% CO 2 Af...

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Abstract

The invention discloses preparation and application of an interferon-polymer combination IFN-PMPC. The invention provides a method for preparing a protein-polymer combination. The method comprises the following step: subjecting a protein-initiator combination, 2-methacryloyloxyethyl phosphorylcholine, CuCl, CuCl2, 1,1,4,7,10,10-hexamethyltriethylene tetramine to a polymerization reaction in a buffer solution, so as to obtain the protein-polymer combination, wherein the protein-initiator combination is a product of covalent connection between an initiator and protein. Proven by experiments, the combination IFN-PMPC prepared by the method is high in yield, simple in purification, specific modified in locus and free of immunogenicity, so that in-vitro biological activity can be better reserved, pharmacokinetics and biological distribution are greatly improved, and the treatment effect is effectively enhanced.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the preparation and application of an interferon polymer conjugate IFN-PMPC. Background technique [0002] Proteins have been widely used in many fields such as biomedical imaging, targeted therapy, and clinical diagnosis. Using protein alone has problems such as short half-life and poor stability. Linking proteins with polymers to prepare protein-polymer conjugates can effectively improve the solubility, stability, pharmacokinetics and therapeutic efficacy of proteins and reduce their immunogenicity. The traditional synthesis method of protein-polymer conjugates is to connect pre-prepared polymers to proteins, which often has uncertain coupling sites, low efficiency, poor yield, difficult separation of products, poor quality control, and difficult activity. Maintain and many other problems. [0003] Interferon-α2 (IFN-α2), a potent inhibitor of viral replication and tum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/555C07K14/56C07K14/565C07K14/57C07K1/107A61K38/21A61K47/59A61K47/58A61P35/00A61P31/12
CPCA61K38/00C07K14/555C07K14/56C07K14/565C07K14/57C07K2319/00
Inventor 高卫平胡瑾王贵林
Owner TSINGHUA UNIV