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Fusion protein of tnfα and dc-sign and its application

A technology of fusion protein and function, which is applied in the fusion protein of TNFα and DC-SIGN and its application field, which can solve the problems of single recognition site, affecting the overall function of monoclonal antibody, and poor binding ability

Active Publication Date: 2016-06-01
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the single-chain antibody has a complex structure, poor binding ability, unstable expression, and a single recognition site. If the antigenic site is mutated, the targeting effect cannot be achieved, which affects the overall function of the monoclonal antibody.

Method used

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  • Fusion protein of tnfα and dc-sign and its application
  • Fusion protein of tnfα and dc-sign and its application
  • Fusion protein of tnfα and dc-sign and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The gene design of embodiment 1 fusion protein

[0062] 1. Linker peptide screening

[0063] The present invention first selects the linking peptide G10 as the linking peptide between TNFα and DC-SIGN.

[0064] Using SYFPEITHI, MH2pred, and ANNpred software, and using their default parameters for calculation, the T cell immunogenicity scores of G10 are calculated as: 5 (the highest score), 0, 0, and the B cell immunogenicity scores calculated by Bcepred and ABCpred are: 0, 0.51, the T cell and B cell immunogenicity scores calculated after normalization were 0.0 and 0.51, which belonged to weak immunogenicity.

[0065] 2. Protein model construction

[0066] The present invention preferably adopts DC-SIGN as the recognition functional domain, and the GenBank number corresponding to DC-SIGN is M98457, and the corresponding protein number is AAF77072 ( figure 1), the full name of DC-SIGN is the specific intercellular adhesion factor 3 binding non-integrin molecule on the...

Embodiment 2A

[0075] The gene optimization of embodiment 2AT3002 fusion protein

[0076] 1. Codon optimization

[0077] In order to better express the AT3002 fusion protein, the applicant also carried out codon optimization, mRNA structure correction and translation initiation site optimization. The obtained gene sequence is shown in SEQ ID NO:1. Gene comparison before and after optimization such as figure 1 shown.

[0078] The following is a comparison of the parameters before and after the codon optimization of the AT3002 gene:

[0079] 1. Codon Adaptation Index (CodonAdaptationIndex, CAI)

[0080] Depend on Image 6 -a shows that before the codon optimization, the codon adaptation index (CAI) of the AT3002 gene in mammalian cells is 0.84. Depend on Image 6 -b It can be seen that after codon optimization, the CAI index of the AT3002 gene of the present invention in mammalian cells is 0.85. Usually, when CAI=1, it is considered that the gene is in the most ideal high-efficiency ex...

Embodiment 3A

[0089] Gene synthesis and expression vector construction of embodiment 3AT3002 fusion protein

[0090] The above-mentioned optimized AT3002 fusion protein gene was artificially synthesized, and HindIII and BamHI restriction sites were added to the two ends of the fusion protein gene. A stop codon site was also introduced behind the BamHI site to prevent the expression of the FLAG tag on the expression vector from affecting the properties of the fusion protein. The fusion protein gene was inserted into the p3xFLAG-CMV-13 plasmid ( Figure 9 a, The plasmid was purchased from Sigma), and a long-term storage plasmid was obtained, which was designated as p3xFLAG-CMV-13-AT3002 plasmid.

[0091] The whole gene of the above-mentioned optimized AT3002 fusion protein was artificially synthesized, and restriction sites of AvrⅡ and BstZ171 were added to the two ends of the full length of the fusion protein gene respectively. The fusion protein gene was inserted into the pCHO1.0 plasmid ...

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Abstract

The present invention relates to the fusion protein of TNFα and DC-SIGN and its application. Traditional anti-tumor drugs or treatment methods tend to cause damage to normal cells in the process of killing tumor cells. The targeted anti-tumor fusion protein of the present invention consists of a recognition functional domain, an action functional domain, and a connecting peptide connecting the two functional domains. The recognition functional domain is a lectin polypeptide with the function of recognizing and binding sugar chains on the surface of tumor cells-specific intercellular adhesion factor 3 on the surface of DCs binding to non-integrin molecules (DC-SIGN, dendritic-cell-specific? ICAM- 3-grabbing? nonintegrin), located at the C-terminus of the fusion protein; the functional domain is tumor necrosis factor (TNF), located at the N-terminus of the fusion protein; the connecting peptide is a short peptide containing 8-25 amino acids. The fusion protein can use lectin to carry out targeted combination with cancer cells, induce apoptosis of cancer cells through pro-apoptotic ligands, and at the same time reduce the killing effect on normal tissues and cells.

Description

technical field [0001] The present invention relates to the field of genetic engineering, and relates to the fusion protein of TNFα and DC-SIGN and its application. More specifically, the present invention relates to the binding and non-integration of specific intercellular adhesion factor 3 on the surface of DCs containing recognition variation or highly expressed sugar chains A fusion protein of the extracellular region domain of DC-SIGN, dendritic-cell-specific ICAM-3-grabbingnonintegrin, connecting peptide and tumor necrosis factor (TNF). Background technique [0002] With the change of people's living environment and lifestyle, the anti-tumor market is gradually expanding. At present, conventional methods such as radiotherapy and chemotherapy for the treatment of tumors cannot distinguish tumor cells from normal tissue cells, often leading to serious side effects, often killing tumor cells at the same time. It also destroys the normal immune function of the body, greatl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K38/19A61K47/48A61P35/00
CPCA61K38/00A61P35/00C07K14/4726C07K14/525C07K2319/00
Inventor 马永侯景姚翔罗成徐春林陈晨王耀方
Owner ZONHON BIOPHARMA INST
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