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Molecule and chimeric molecules thereof

A chimeric molecule and protein technology, applied in cytokines/lymphokines/interferons, drug combinations, peptide/protein components, etc., can solve the problems of pollution, unsuitable for clinical application, and harmful to transplantation applications.

Inactive Publication Date: 2010-12-29
APOLLO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, stem cells are routinely maintained in media that include non-human proteins, which is not suitable for clinical use due to the possibility of contamination with non-human infectious substances
Further, stem cell culture in non-human derived media may result in the introduction of non-human carbohydrate moieties thus compromising transplantation applications

Method used

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  • Molecule and chimeric molecules thereof
  • Molecule and chimeric molecules thereof
  • Molecule and chimeric molecules thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1451] (a) Preparation of pIRESbleo3-Fc construct

[1452] The DNA sequence encoding the Fc domain of human IgG1 was amplified by polymerase chain reaction (PCR), from the EST cDNA library (Clone ID 6277773, Invitrogen), using restriction enzymes BamH1 and BstX1 sites incorporated respectively Forward primer (SEQ ID NO: 21) and reverse primer (SEQ ID NO: 22). This amplicon was cloned into the corresponding restriction site in pI RESbleo3 (Cat. No. 6989-1, BD Biosciences) to prepare the construct pIRESbleo3-Fc. pIRESbleo3-Fc was digested with BamH1 and BstX1 to release an insert of the desired size of 780bp, as determined by gel electrophoresis.

[1453] (b) Preparation of DNA construct expressing protein

[1454] The DNA sequence encoding the protein was amplified by PCR, from the EST cDNA library, using a forward primer and a reverse primer that introduced restriction sites according to Table 8. After amplification, the amplicon was digested with appropriate restriction enzymes a...

Embodiment 2

[1462] (a) Production and purification of human cells expressing GM-CSF

[1463] (i) Production of GM-CSF of the present invention

[1464] On day 0, 3×10 cells from the transformed embryonic human kidney cell line 7 Cell seeding five 500cm 2 Tissue culture plates (Corning), such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen). Cells were inoculated into 90ml Dulbecco's modified Eagle's medium / Ham's nutrient mixture F12 (DMEM / F12) (JRH Biosciences) per plate. The medium was supplemented with 10% (v / v) donor calf serum (FCS, JRH) Biosciences), 10 mM HEPES (Sigma), 4 mM L-glutamine (Ameresco) and 1% (v / v) penicillin-streptomycin (JRH Biosciences). In addition, 10% (v / v) heat-inactivated donor bovine serum (DCS, JRH Biosciences) can be used to replace FCS without HEPES.

[1465] On day 1, transfection was performed with calcium phosphate. Before transfection, each culture plate was replaced with 120ml of newly prepa...

Embodiment 3

[1523] (a) Characterization of GM-CSF of the present invention

[1524] (i) Two-dimensional polyacrylamide electrophoresis

[1525] The sample collected in Example 2(a) was passed through a dialysis or desalting column (Pharmacia HR10 / 10 Fast Desalting Column) to replace the buffer into repurified (18 MOhm) water, and dried with a SpeedVac concentrator. In addition, the latest TCA or acetone precipitation technology can be used to process the collected samples. Then, the sample was dissolved in 240ml MSD buffer (5M urea, 2M thiourea, 65mM DTT, 2% (w / v) CHAPS, 2% (w / v) thiobetaine 3-10, 0.2% (v / v) Carrier ampholyte, 40mM Tris, 0.002% (w / v) bromophenol blue, water) and centrifuged at 15000g for 8 minutes.

[1526] Isoelectric focusing (IEF) was performed with precast 11cm or precast 17cm gel pH3-10 solid phase pH gradient IEF strips (BioRad). The IEF strips are rehydrated in the sample in the closed tube at room temperature for at least 6 hours. The IEF strip is placed in the focu...

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Abstract

The present invention relates generally to the fields of proteins, diagnostics, therapeutics and nutrition. More particularly, the present invention provides an isolated protein molecule in or related to the short chain 4 helix bundle superfamily such as GM-CSF, IL-3, IL-4 and IL-5 or chimeric molecules thereof comprising at least a portion of the protein molecule, such as GM-CSF-Fc, IL-3-Fc, IL-4-Fc and IL-5-Fc; wherein the protein or chimeric molecule thereof has a profile of measurable physiochemical parameters, wherein the profile is indicative of, associated with or forms the basis of one or more pharmacological traits. The present invention further contemplates the use of the isolated protein or chimeric molecule thereof in a range of diagnostic, prophylactic, therapeutic, nutritional and / or research applications.

Description

Technical field [0001] The present invention generally relates to proteinology, diagnostics, therapeutics and nutrition. In particular, the present invention provides an isolated protein molecule, which belongs to the 4helix bundle superfamily or is related to the 4helix bundle superfamily, such as: GM-CSF, IL-3, IL- 4 and IL-5 and chimeric molecules containing at least a part of the protein molecule, for example: GM-CSF-Fc, IL-3-Fc, IL-4-Fc and IL-5-Fc. The protein or its chimeric molecule has a measurable parameter characteristic, wherein the characteristic represents, correlates with, or forms the basis of one or more pharmacological properties. The present invention further envisages the use of the isolated protein or its chimeric molecule in the scope of diagnosis, prevention, treatment, nutrition and / or research applications. Background technique [0002] Any prior art referred to in this specification is not, and should not be understood as, an approval or any form of re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/535A61K38/20A61K38/19C07K14/54A61P37/02
Inventor J·D·普里斯特A·D·沃茨J·S·惠特克G·R·皮尔金顿C·A·利德尔I·贝姆
Owner APOLLO LIFE SCI