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A series of fusion protein with dual biological activity and medical treatment thereof

A technology that fuses proteins and sequences, applied in the fields of genetic engineering and protein engineering, and can solve problems such as the lack of treatment methods for patients

Active Publication Date: 2008-04-23
INNOVENT BIOLOGICS (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existing TNFα inhibitors have achieved great success, they are only effective for 40% of patients, and most of the remaining patients lack effective treatments, so the development of a new generation of anti-inflammatory products is of great significance

Method used

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  • A series of fusion protein with dual biological activity and medical treatment thereof
  • A series of fusion protein with dual biological activity and medical treatment thereof
  • A series of fusion protein with dual biological activity and medical treatment thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Cloning of DNA sequences encoding dual-active fusion proteins and construction of recombinant vectors

[0073] The coding DNA of the dual-activity fusion protein in the present invention is obtained by amplifying cDNA of TNFR2, IL-6 binding peptide M123, and immunoglobulin IgG1Fc with different primers by polymerase chain reaction (PCR).

example 1

[0074] Example 1 Construction of TG-001 gene and recombinant vector

[0075] TG-001 is fused by the extracellular fragment P75 of TNFR2 and human immunoglobulin Fc. Its N-terminal contains the signal peptide of TNFR2 to ensure its extracellular secretion. There is a 12 amino acid (G12) between P75 and Fc. ) of the peptide linker.

[0076] Wherein the PCR fragment (824bp) of P75 is obtained by amplifying the plasmid pORF9-hTNFRSF1B(s) (Invivogen Company) as a template and the following primers:

[0077] Primer 1: 5'-GGCTAGCCTCGAGAATTCGCAACCACCATGGCGCCCGTCGCCGTCTG-3'

[0078] Primer 2: 5'-CCACCTCCGCCACCTCCGCCTCCACCGTCGCCAGTGCTCCCTTCAG-3'

[0079] The PCR fragment (724bp) of Fc is amplified from human lymph node cDNA (BD Clontech Company) as a template and the following primers:

[0080] Primer 3: 5'-CTATCTCACACATCGACAATTCGAAGACAAAACTCACACATGCCCAC-3'

[0081] Primer 4: 5'-AAGGGAATCTAGAGCGGCCGCTCATTTACCCGGAGACAGGGAG-3'

[0082] Then the fusion fragment (1539bp) of P75-Fc wa...

example 2

[0084] Example 2 Construction of TG-002 gene and recombinant vector

[0085] TG-002 is fused by the extracellular fragment P75 of TNFR2, the peptide chain PEP17 in the α chain of GM-CSF receptor, and human immunoglobulin Fc, and its N-terminus contains the signal peptide of TNFR2 to ensure its extracellular secretion , between P75 and PEP17, and between PEP17 and Fc, there is a peptide linker segment containing 6 glycines. The construction of TG-002 is based on the TG-001 plasmid as a template and constructed by splicing PCR (Bridge PCR).

[0086] The construction of the TG-002 coding DNA first PCR-amplified the TG-001 plasmid with primers 5 and 6, and primers 8 and 9 to obtain two PCR products (168bp and 273bp), and then used them as templates and carried out with primers 1 and 6. The result of splicing PCR (421bp).

[0087] Primer 5: 5'-TCCACACGATCCCAACACAC-3'

[0088] Primer 6: 5'-GTCCAGGTACGACAGCTTCTGATAGGTCCTGGGCTGTTTGCCACCTCCGCCTCCACCGTC-3'

[0089] Primer 7: 5'-AG...

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Abstract

The present invention is series of innovative fusion proteins with double bioactivity. They can block tumor necrosis factor and block granulocyte-macrophage colony stimulating factor or interleukin-6 simultaneously, and has high bioactivity, excellent stability, proper in vivo half-life. They may be applied in inhibiting inflammation effectively and treating autoimmune diseases. The present invention includes these fusion proteins, and their coding recombinant DNA sequence, expression vector, medicinal use, medicines and preparation.

Description

technical field [0001] The present invention relates to the technical fields of genetic engineering and protein engineering, in particular to a DNA sequence encoding a dual biologically active fusion protein capable of simultaneously inhibiting tumor necrosis factor and granulocyte-macrophage colony-stimulating factor or interleukin-6, and its The encoded fusion protein, the pharmaceutical use of the fusion protein, the medicine containing the fusion protein and its preparation. Background technique [0002] Tumor necrosis factor α (TNFα for short) is a multifunctional cytokine that plays an important role in biological mechanisms such as inflammatory response, immune regulation, and anti-infection (Aggarwal, Nature Reviews Immunology, 2003, 3, 745 -756). TNFα has two widely expressed cell surface receptors: TNFR1 (also known as p55 or CD120a) and TNFR2 (also known as p75 or CD120b). The combination of TNFα and TNFR1 or TNFR2 can trigger a series of biological responses, in...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/16A61P31/00A61P37/00
Inventor 余波郑佳
Owner INNOVENT BIOLOGICS (SUZHOU) CO LTD
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