Molecules and chimeric molecules thereof

A technology of chimeric molecules and compositions, applied in the direction of cytokines/lymphokines/interferons, hybrid peptides, cytokines/lymphokines/interferon receptors, etc., can solve inapplicable clinical applications, pollution, hazards Porting applications and other issues

Inactive Publication Date: 2008-04-09
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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  • Molecules and chimeric molecules thereof
  • Molecules and chimeric molecules thereof
  • Molecules and chimeric molecules thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1342] Preparation of vector-Fc construct

[1343] (a)pIRESbleo3-Fc

[1344] 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). The amplicon was cloned into the corresponding restriction site in pIRESbleo3 (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.

[1345] (b) Preparation of DNA construct expressing protein or protein-Fc

[1346] The DNA sequence encoding the protein or its extracellular domain was amplified by PCR, from the EST cDNA library, using forward and reverse primers with restriction enzyme cleavage sites introduced according to Table 8. After amplification, ...

Embodiment 2

[1356] (a) Preparation, separation and purification of TNF-α of the present invention

[1357] (i) Preparation of TNF-α of the present invention

[1358] On day 0, at 5 500cm 2 Spread 3×10 on the tissue culture dish (Corning) 7 Cells of a transformed human embryonic kidney cell line, such as HEK 293, HEK293cl8, HEK 293T, 293CEN4, HEK 239F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen). To each culture dish, add 90ml Dulbecco's Modified Eagle's Medium / Ham Nutritional Complex F12 (DMEM / F12) (JRH Biosciences) to the cells, and add 10% (v / v) heat-inactivated fetal calf serum (FCS, JRH Biosciences) to the medium ), 4mM L-glutamine (Amresco), 10mM HEPES (Sigma), and 1% (v / v) penicillin-streptomycin (penicillin G 5000U / ml, streptomycin sulfate 5mg / ml) (JRHBiosciences). Petri dish at 37°C and 5% CO 2 Incubate overnight under the appropriate conditions.

[1359] On day 1, transfection was performed with calcium phosphate. Before transfection, the medium in each plate was re...

Embodiment 3

[1465] (a) Characterization of TNF-α of the present invention

[1466] (i) Two-dimensional polyacrylamide electrophoresis

[1467] Example 2(a) The collected sample was passed through a dialysis or desalting column (Pharmacia HR 10 / 10 Fast Desalting Column) to replace its buffer with repurified (18 MOhm) water, and dried with a SpeedVac concentrator. Alternatively, the collected samples can be precipitated with TCA or acetone using a known method. The sample was dissolved in 240μl of MSD buffer (5M urea, 2M thiourea, 65mM DTT, 2% (w / v) CHAPS, 2% (w / v) sultaine 3-10, 0.2% (v / v) Ampholyte carrier, 40mM Tris, 0.002% (w / v) bromophenol blue, water), and centrifuged at 15000g for 8 minutes.

[1468] Isoelectric focusing (IEF) was performed with precast 11cm or precast 17cm gel pH 3-10 solid phase pH gradient I EF 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 focusing chamber and c...

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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 tumour necrosis factor (TNF) superfamily such as TNF-a, Lymphotoxin-a (LT-a), TNFRI, TNFRII, OX40, BAFF, NGFR, Fas Ligand or chimeric molecules thereof comprising at least a portion of the protein molecule, such as TNF-a-Fc, LT-a-Fc, TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, Fas Ligand-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 belonging to or related to the tumor necrosis factor (TNF) superfamily and a chimeric molecule comprising at least a part of the protein molecule, the isolated protein, such as TNF-a, Lymphotoxin-a (LT-a), TNFRI, TNFRII, OX40, BAFF, NGFR, Fas ligand; the chimeric molecule comprising at least a part of the protein molecule, such as TNF-a-Fc, LT-a-Fc , TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, Fas ligand-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 diagnostic, prophylactic, therapeutic, nutritional and / or research appli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/525A61K38/17A61K38/19A61K39/395A61K45/00A61P17/00C07H21/04C07K14/715C07K19/00G01N33/543
Inventor J·D·普里斯特A·D·沃茨J·S·惠特克T·A·多马加拉G·R·皮尔金顿I·贝姆C·M·Y·李林美安N·S·托马斯
Owner APOLLO LIFE SCI
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