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PEG interleukin 15

A technology of human interleukin and conjugates, applied in the fields of peptide/protein components, chemical instruments and methods, medical preparations of non-active ingredients, etc., can solve problems such as no research

Active Publication Date: 2012-09-26
BEIJING KAWIN TECH SHARE HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

PEG-conjugated polymers of interleukin 15, no related research so far

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of rhIL-15

[0046] Test materials: DNA restriction endonuclease, T4DNA ligase, TaqDNA polymerase, and M-MLV reverse transcriptase are all products of GIBCO; Escherichia coli host strain DH5a, BL21 (DE3) (purchased from Invotrogen); pVB220 vector (purchased from Novagen)

[0047] 1 Design and synthesis of primers: design a pair of primers according to the gene sequence of hIL1-15 reported, upstream primer P1: 5'-C ctcgagTTCATGAACTGGGTGAATGTAATAAG-3', including the start codon ATG, peripheral xho I restriction site; downstream primer P2: 5'-CGGATCCttaTTAAGAAGTGTTGATGAAGATTTG-3', change the stop codon into a strong terminator TAA frequently used in prokaryotic systems, peripheral Bam H I restriction site, primers were synthesized by Beijing Sanbo Polygala Biotechnology Co., Ltd.

[0048] 2 Preparation of mononuclear cells rich in rhIL-15mRNA and extraction of total RNA, normal human peripheral blood leukocytes were separated by conventional dens...

example 2

[0052] Example 2, recombinant integrated interferon variant PEGylated conjugate (mPEG 20 -rhIL-15) modification conditions

[0053] rhIL-15 solution (rhIL-15, concentration 10 mg / mL, buffered in 100 mM PB, pH 7.0) was prepared by Beijing Kain Technology Co., Ltd., mPEG-ALD-20KD (purity>95%, relative molecular weight Mass is 20×10 3 ) was purchased from Beijing Kaizheng Bioengineering Development Co., Ltd., sodium cyanoborohydride (CH3BrNa) solution was purchased from Sigma Company, SDS-PAGE related reagents and solutions and various analytical grades were from Shanghai Sangon. Constant temperature magnetic stirrer (Jiangsu Zhongda Instrument Factory), electrophoresis instrument, Q Sepharose F.F., Superdex75 molecular sieve, AKTA explore chromatography system, and SDS-PAGE electrophoresis system are all products of Pharmacia.

[0054] 1. The influence of reaction pH value on the modified product

[0055] The rhIL-15 solution was made up to 2 mg / mL with 100 mmol / L PB (p...

Embodiment 3

[0065] Example 3 Purification of mPEG-rhIL-15

[0066] The reaction mixture was first dialyzed against 50mM Tris-HCl buffer (pH 6.5), and then the sample was loaded on a Q Sepharose FF cation exchange column equilibrated with the same buffer. The adsorbed protein was eluted with a 0-1M NaCl gradient. PEG-interferon was further purified by Superdex 75, and the mobile phase was 50mM Tris-HCl buffer (pH 6.5) containing 0.15M sodium chloride. The eluted active fractions were collected, and the SDS-PAGE test results showed that PEG-interleukin had been purified to a single electrophoresis band.

[0067] The process of coupling rhIL-15 with 10kDa and 40kDa is the same as above, and the electrophoresis of the purified PEG interleukin is a single band, see the attached image 3 .

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Abstract

The invention relates to a PEG interleukin 15 conjugate, namely PEG interleukin 15. PEG and interleukin 15 are connected on an N terminal amino group through or not through small peptide connexon to obtain the uniform PEG interleukin 15 conjugate. The activity of the original interleukin 15 can be at least reserved in vivo, and the PEG interleukin 15 conjugate has longer half-life period and average time to peak.

Description

technical field [0001] The invention relates to a chemically modified polyethylene glycol interleukin-15 conjugate, which belongs to the field of biopharmaceuticals. Background technique [0002] Interleukin 15 (IL-15) is a cytokine of about 12-14kD discovered by Grabstein et al. in 1994. The natural human interleukin 15 mature peptide contains 114 amino acids, including 4 cysteine ​​residues, Cys35 is connected with Cys85, Cys42 and Cys88, and the two pairs of intramolecular disulfide bonds formed play an important role in maintaining the spatial conformation and biological activity of IL-15. [0003] Similar to most cytokines, IL-15 can play a role in the normal immune response of the body, such as promoting the development of T cells, B cells, natural killer (NK) cells, etc. Various tissues and cells play a broad and pleiotropic role. In vitro studies have shown that IL-15 and IL-2 share several biological activities due to shared receptor components. The IL-15 receptor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/48A61K38/20C07K17/08A61P35/00A61K47/60A61K47/65
Inventor 侯建华张春丽杨璐
Owner BEIJING KAWIN TECH SHARE HLDG
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