TNF alpha and DC-SIGN fusion protein and application thereof

A fusion protein and function technology, applied in the fusion protein of TNFα and DC-SIGN and its application field, can solve the problems of inability to achieve targeting, single recognition site, affecting the overall function of monoclonal antibody, etc.

Active Publication Date: 2014-04-23
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. I

Method used

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  • TNF alpha and DC-SIGN fusion protein and application thereof
  • TNF alpha and DC-SIGN fusion protein and application thereof
  • TNF alpha and DC-SIGN fusion protein and application thereof

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 (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 expression state in the ...

Embodiment 3A

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

[0090] The above-mentioned optimized full gene of AT3002 fusion protein was artificially synthesized, and restriction sites of Hind III and BamH I were respectively added to the two ends of the full length of the fusion protein gene. A stop codon site was also introduced behind the BamH I 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 prote...

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Abstract

The invention relates to a TNF alpha and DC-SIGN fusion protein and an application thereof. In a traditional anti-tumor medicament or a treatment method, normal cells are easy to damage in the process of killing tumor cells. The targeting anti-tumor fusion protein provided by the invention comprises an identification functional domain and an action functional domain, as well as a connecting peptide for connecting the two functional domains. The identification functional domain is as follows: a lectin polypeptide with tumor cell surface sugar chain identification and combination functions-a DC surface specific intercellular adhesion factor 3 combined with non-integrin molecules (DC-SIGN, dendritic-cell-specific ICAM-3-grabbing nonintegrin) is positioned at a C terminal of the fusion protein; the action functional domain is as follows: a tumor necrosis factor (TNF) is positioned at an N terminal of the fusion protein; and the connecting peptide is a short peptide containing 8-25 amino acids. The fusion protein can utilize lectin and cancer cells to perform targeting combination, induce the apoptosis of the cancer cells through a ligand capable of promoting the apoptosis of the cells and simultaneously reduce the killing effect against 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 dendritic-cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN, dendritic-cell-specific ICAM-3-grabbing nonintegrin), 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 ...

Claims

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Application Information

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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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