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Recombinant IL-15 fusion protein and application thereof

A fusion protein, albumin nanotechnology, applied in the direction of fusion polypeptides, drug combinations, immunoglobulins, etc., can solve the problems of PEGylation of target proteins, high requirements, and decreased protein activity, and achieve the effect of preventing and treating tumors.

Active Publication Date: 2020-08-11
SUZHOU PRO HEAL PHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PEGylation has high requirements on the process, and at the same time, PEGylation has problems such as the inability to PEGylate the target protein at a fixed point, and the activity of the protein after PEGylation decreases.

Method used

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  • Recombinant IL-15 fusion protein and application thereof
  • Recombinant IL-15 fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Gene synthesis and vector construction of recombinant IL-15 fusion protein

[0046] IL-15 (SEQ ID NO: 1), IL-15Rα Sushi (SEQ ID NO: 2 ), the amino acid sequence of anti-HSA VHH (SEQ ID NO: 4) was assembled, and IL-15 and IL-15RαSushi were indirectly connected through a flexible linker peptide (SEQ ID NO: 3) to form a recombinant IL-15 fusion protein sequence (SEQ ID NO: 5), according to the codon optimization of human host cells and routinely synthesized genes, and then add an EcoRI restriction site and 5' UTR at the 5' end in sequence ([SEQ ID NO: 6]); at the 3' end The 3'UTR ([TGATGA]) and HindIII restriction sites were added in turn, and the gene was cloned into the vector pTT5 (SEQ ID NO: 7) through 5' EcoRI and 3' HindIII. Select the clones for sequencing, select the correctly sequenced bacteria for preservation and expand the culture of the bacteria, and the expanded bacteria are used for plasmid extraction.

Embodiment 2

[0047] Example 2 Expression and purification of recombinant IL-15 fusion protein

[0048] Perform cell transfection and protein separation and purification on the extracted plasmids as follows:

[0049] 1. Measure the cell density. The viability should be greater than 95%. Use the preheated 293 medium to adjust the 293 cell density to 3×10 6 Cells / mL, shake gently and distribute the cells (transfection system 90%), the volume of cells in the shake flask should not exceed 1 / 3 of the shake flask specification, and put it in a shaker for use.

[0050] 2. Calculate the volume of transfection buffer opti-MEM according to the volume of transfected cells, which is 1 / 10 of the transfection system; calculate the amount of transfection reagent PEI, whose ratio is 3 μL / mL transfected cells; calculate the volume of transfected DNA The total amount, the ratio is 1 μg / mL transfected cells.

[0051] The specific transfection procedure is as follows:

[0052] Take a 50mL centrifuge tube, a...

Embodiment 3

[0057] Example 3 Binding detection of recombinant IL-15 fusion protein and HSA

[0058] Using Gator TM A non-labeled bioanalyzer was used to detect the affinity of the recombinant IL-15 fusion protein expressed above and HSA protein (purchased from ACROBiosystems, catalog number HSA-H5220), and the Protein A biosensor was used to capture the antibody sample, and then the captured recombinant IL-15 15 The kinetic detection of the binding and dissociation of the fusion protein and the HSA protein. Kinetics were analyzed by fit using a 1:1 binding model. The brief steps are as follows: protein loading for 200s; binding for 180s; dissociation for 300s; regeneration for 30s.

[0059] Use Gator TM The affinity data determined by the instrument are shown in Table 1 below.

[0060] Table 1 Affinity test results of recombinant IL-15 fusion protein and HSA protein

[0061] antigen concentration fusion protein Response Affinity (M) Dissociation constant (1 / s) B...

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Abstract

The invention belongs to the technical field of medicines, and particularly relates to a recombinant IL-15 fusion protein and application thereof. The recombinant IL-15 fusion protein provided by theinvention comprises interleukin-15, a Sushi structural domain of an interleukin-15 receptor alpha and an anti-human serum albumin nano antibody, wherein the interleukin-15, the Sushi structural domainof the interleukin-15 receptor alpha and the anti-human serum albumin nano antibody are directly connected or indirectly connected through connecting peptides. The recombinant IL-15 fusion protein can be combined with human serum albumin, and T cells can be effectively activated at a low dose concentration, so that an immune system plays a role, and the purposes of preventing and treating tumorsare achieved.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a recombinant IL-15 fusion protein and its application. Background technique [0002] Cytokines, such as lymphokines, chemokines and growth factors, are produced by various types of cells including lymphocytes and monocytes, and play a wide range of roles in human physiology and disease control. These types of cytokines have been clinically proven to be effective in the treatment of various diseases. For example, interleukin (interleukin, IL) is a kind of cytokine produced by various cells and acts on various cells. It transmits information, activates and regulates immune cells, and mediates the activation, proliferation and differentiation of T and B cells. Plays an important role in the inflammatory response. [0003] Among them, interleukin-15 (IL-15) belongs to a family of four small α-helical bundle cytokines. IL-15 exerts its biological activity through binding to its r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85C12N5/10A61K38/20A61K38/17A61K39/395A61P35/00A61P35/02
CPCA61K38/1793A61K38/2086A61K39/39558A61P35/00A61P35/02C07K14/5443C07K14/7155C07K16/18C07K2317/569C07K2319/00C12N15/85A61K2300/00
Inventor 张鹏马琳
Owner SUZHOU PRO HEAL PHARM TECH CO LTD
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