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Long-acting recombinant human interleukin 2 fusion protein and preparation method and application thereof

A technology of interleukin and fusion protein, which is applied in the field of biopharmaceuticals, can solve the problems of increasing side effects, increasing treatment costs, increasing patient pain, etc., and achieves the effects of promoting proliferation, prolonging plasma half-life, and high expression

Inactive Publication Date: 2021-12-14
BEIJING VDJBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned diseases generally have a long course of disease, so long-term use of IL-2 is required, and the plasma half-life of IL-2 is only about 2 hours. In order to maintain the efficacy of the drug, frequent injections of large doses are required, which not only greatly increases the cost of treatment, but also increases the cost of treatment. Patient suffering, increased side effects

Method used

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  • Long-acting recombinant human interleukin 2 fusion protein and preparation method and application thereof
  • Long-acting recombinant human interleukin 2 fusion protein and preparation method and application thereof
  • Long-acting recombinant human interleukin 2 fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Constructing a Stably Transduced Cell Line

[0045] The day before transfection, adjust the density of CHO-K1 cells to 0.5×10 6 cells / mL. On the day of transfection, prepare a linearized, high-concentration endotoxin-free plasmid, measure the CHO-K1 cell density and viability, and ensure that the cell viability is greater than 97%. After CHO-K1 cells were washed twice with CD CHO medium, configure the electroporation reaction system: 700 μL cell suspension + 40 μg plasmid, mix well and transfer to a 4 mm electrode cup. Put the electrode cup into the electroporator, set the electric shock parameters to 300V, 1000μF, electric shock once, transfer the cell suspension after electric shock into the preheated fresh CD CHO medium, and incubate at 37°C for 20min. The incubated cell suspension was uniformly inoculated in a 96-well plate. After 24 hours of transfection, the pressure was applied, and CD CHO medium containing methionine iminosulfone (MSX) was added. The...

Embodiment 2

[0046] Example 2 Screening high expression monoclonal cell lines

[0047] After the single clones in the 96-well plate grow to a suitable size, start to pick the single clones, and transfer all the clones to a new 96-well plate, 5% CO 2 , 37 ℃ static culture. After the cells in the wells were overgrown, the supernatant in the well plate was taken for reduction electrophoresis to detect the expression of the fusion protein, and the 9 clones with the highest expression were selected (see figure 1 ), gradually expanded to shake flask culture. Nine clones were fed-batch cultured in 25mL shake flasks, and the culture supernatant was harvested for identification by non-reducing electrophoresis (see figure 2 ), the cell line #9 with the best expression was selected. Cell line #9 was screened for monoclonal cell lines by the limiting dilution method, and 0.3 cells / well were inoculated in a 96-well plate, and 11 high-expressing cell lines were screened, and fed-batch cultured in a ...

Embodiment 3

[0048] 125mL shake flask fed-batch culture of stable cell line in embodiment 3

[0049] On the day when IL-2-HSA / CHOK1 cells begin to be cultured and expressed, press 0.3×10 6 Inoculate 25mL of basal medium containing 25-50μM MSX into a 125mL shaker flask per cell / mL, record it as D0 at this time, 5% CO 2 , 37°C, 135rpm shaker culture. Inoculate D4 to start sampling and counting, and the cell density reaches 10×10 6 1 cells / mL, the culture temperature was lowered to 33°C. D5 began to feed the feed medium, and control the glucose concentration to 3-4g / L. On culture D13, the culture was terminated, the supernatant of the cell culture fluid was collected, and the expression level of the fusion protein was determined to be 4.36 mg / mL.

[0050] For the kinetic curve of IL-2-HSA / CHOK1 cell culture, see Figure 5 . Depend on Figure 5 It can be seen that in the early stage of culture, the cells are in the logarithmic growth stage, and the density increases rapidly; in the late...

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Abstract

The invention discloses a fusion protein of interleukin 2 and human serum albumin. Plasmids with a fusion gene of interleukin 2 and human serum albumin are electrically transferred into CHO cells to obtain a CHO monoclonal cell strain capable of stably and efficiently expressing human recombinant protein. The monoclonal cell strain of the invention can secrete and express the fusion protein of the interleukin 2 and the human serum albumin, and the fusion protein can prolong the plasma half-life period of the human interleukin 2 and can be used for preparing human interleukin 2 drugs or drugs for treating various diseases, such as tumors and immunodeficiency diseases.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a fusion protein of human interleukin 2 and human serum albumin, its encoding gene and its application. Background technique [0002] Interleukin 2 (IL-2) is a 15.5kD glycoprotein produced by T cells and NK cells, which plays an important role in the body's immune response. (Human) Interleukin 2 ((h)IL-2) consists of 133 Amino acid residue composition (SEQ ID NO: 1), with an intrachain disulfide bond and a cysteine ​​located at the 125th amino acid residue of the mature protein, this cysteine ​​is easily bonded to the other two cysteines Mismatched disulfide bonds are formed, and IL-2 with abnormally paired disulfide bonds has no activity. Mutating the cysteine ​​to a serine or alanine avoids this problem without altering its activity. IL-2 mainly acts on immune cells, including T cells, large granular lymphocytes, monocytes, and B cells, to promote cell proliferation and secr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K19/00C12N15/62A61K38/20A61K47/64A61P35/00A61P37/02A61P1/16A61P11/00A61P29/00
CPCC12N15/85C07K14/55C07K14/765A61K38/2013A61K47/643A61P35/00A61P37/02A61P1/16A61P11/00A61P29/00C07K2319/02C07K2319/31C12N2800/107C12N2800/22C07K14/76C07K14/47A61P37/00
Inventor 李自强田新生成健伟马荣姜彦静孙艺萍张小锐赵婷婷张筠
Owner BEIJING VDJBIO
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