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IL-2 receptor complex and its preparation method and application

A technology of IL-2, 1. IL-2, applied in the field of genetic engineering, can solve the problems of limitations, inability to directly form high-affinity IL-2 receptor complexes, etc., achieve high-affinity activity, and realize large-scale production and preparation , Highly reproducible effect

Active Publication Date: 2021-06-22
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the absence of IL-2, α, β and γ cannot directly form a high-affinity IL-2 receptor complex
This also limits applications such as antibody preparation against IL-2R αβγ heterotrimers

Method used

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  • IL-2 receptor complex and its preparation method and application
  • IL-2 receptor complex and its preparation method and application
  • IL-2 receptor complex and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of recombinant expression vector

[0058] Construction of the fusion protein I recombinant expression vector pHEK-B1: a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO: 1 was synthesized by Shanghai Bioengineering Co., Ltd. (the amino acid sequence encoding the protein is shown in SEQ ID NO: 2), and in A restriction endonuclease BamH Ⅰ cutting site and a Kozak sequence were added to the 5' end; a stop codon TAA and a restriction endonuclease Xho Ⅰ cutting site were added to the 3' end. The synthesized product was double-digested with restriction endonucleases BamH Ⅰ and Xho Ⅰ, then subjected to agarose gel electrophoresis, and the target fragment was recovered by cutting the gel. The target fragments were respectively combined with pcDNA3.1(+) (Invitrogen Company, catalog number: V790-20) ( figure 2 ) connection to construct the recombinant eukaryotic expression vector pHEK-B1, transform Escherichia coli E.coli DH5α, culture...

Embodiment 2

[0060] Example 2 Recombinant expression vector transfected HEK293 cells and expressed IL-2R αβγ heterotrimeric protein

[0061] Using PEI transfection reagent, the constructed recombinant plasmids pHEK-B1 and pHEK-AG1 were transfected into HEK293 cells at a mass ratio of 1:1 for recombinant expression of heterotrimers. The day before transfection, HEK293 cells were divided into 1 × 10 6 / mL subcultured at 37°C. On the day of transfection, count and adjust the cell density to 1.8×10 6 -2.2×10 6 / ml, the activity rate is over 95%.

[0062] Calculate the amount of each component in the transfection complex according to the cell density: the corresponding relationship between the plasmid dose and the number of cells is 1×10 6 The cells correspond to 1 ug of plasmid, and the corresponding relationship between the dose of PEI and the dose of plasmid is that the quality of PEI is 3 times the quality of DNA.

[0063] Prepare the transfection complex according to the amount of com...

Embodiment 3

[0064] Example 3 Separation and Purification of IL-2R αβγ Heterotrimeric Protein

[0065] Use the method of Protein A affinity chromatography to separate and purify the heterotrimeric recombinant protein prepared in Example 2: 4°C, centrifuge at 1000g for 30 min to collect the cell culture supernatant, filter the supernatant and combine with binding buffer beforehand (20mM Tris-HCl, 150mM NaCl, pH 8.0) equilibrated Protein A chromatography column (Stuofan Bioengineering Technology (Guangzhou) Co., Ltd., catalog number: 17543803), and then washed with elution buffer (100 mM glycine, 150 mM NaCl , pH3.5) to elute the target protein, add 1 / 40 volume of neutralization buffer (2M Tris-HCl, pH8.0) to neutralize after collecting the protein eluate, and perform SDS-PAGE ( image 3 ) and HPLC analysis ( Figure 4 ). According to the results of SDS-PAGE, the purified two bands are located around 66KDa and 116KDa respectively, in line with the theoretical molecular weight of protein (I...

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Abstract

The invention discloses an IL-2 receptor complex and a preparation method and application thereof. The IL-2 receptor complex of the present invention is constructed by constructing nucleic acid sequences encoding fusion protein I (SEQ ID NO: 1 or 7) and fusion protein II (SEQ ID NO: 3 or 9) into the same eukaryotic expression vector or separately Constructed on different eukaryotic expression vectors, transfected mammalian cells with the obtained recombinant expression vector, and separated and purified from the cell culture supernatant. The present invention utilizes genetic engineering technology to directly obtain IL-2 receptor αβγ heterotrimeric protein through recombinant expression and one-step affinity chromatography purification without in vitro assembly; the heterotrimeric protein and IL-2 have high affinity It can be used to study the interaction between IL-2 and IL-2 receptor in vitro, to prepare antibodies from immunized animals or to screen antibodies in vitro.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to IL-2 receptor complex and its preparation method and application. Background technique [0002] Interleukin-2 (IL-2) is one of the earliest discovered cytokines, also known as T cell growth factor (Tcell growth factor, TCGF). In 1976, Robert Gallo of the NCI Tumor Cell Biology Laboratory used PHA to stimulate lymphocyte production conditioned medium to culture bone marrow cells and found that 90% of T cells survived for up to 9 months. Prior to this, primary T cells could not be cultured in vitro, which was a bottleneck technical problem in the development of immunology. The substance that supports the long-term growth of T cells is called T-cell growth factor (TCGF), also known as IL-2. IL-2 plays an important role in the proliferation, development, differentiation and apoptosis of T cells. [0003] IL-2 receptor interleukin-2 receptor (IL-2R) contains three subunits of α, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/62C07K19/00G01N33/68
CPCC07K14/7155C07K2319/00C07K2319/30C07K2319/73C12N15/85C12N2800/107G01N33/6854G01N33/6869
Inventor 秦丽丽陈宜顶苗景赟宋洁
Owner ACROBIOSYSTEMS INC
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