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Fusion protein with double functions of inhibiting revascularization of tumor microenvironment and activating adaptive immune response, and gene and application thereof

A dual-function, gene technology, applied in the field of bioengineering, can solve the problem of not changing the function of T cells

Inactive Publication Date: 2013-07-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Co-stimulatory signals can enhance effector function by increasing antigen specificity and the number of effector CD8+ T cells; however, in the absence of CD3 antibody signal, stimulation of CD137 molecules cannot change T cell function, indicating that CD137 interacts with CD137L to provide co-stimulatory signal

Method used

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  • Fusion protein with double functions of inhibiting revascularization of tumor microenvironment and activating adaptive immune response, and gene and application thereof
  • Fusion protein with double functions of inhibiting revascularization of tumor microenvironment and activating adaptive immune response, and gene and application thereof
  • Fusion protein with double functions of inhibiting revascularization of tumor microenvironment and activating adaptive immune response, and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in E. coli expression system

[0071] 1. Construction of expression system

[0072] A total of 18 bifunctional recombinant proteins with Tumstatin activity and CD137L activity were designed, which were fusions of Tumstatin1 / Tumstatin2 / Tumstatin3 / Tumstatin7 and CD137L1 / CD137L5 / CD137L6 with connecting peptides ( figure 1 ). Among them, CD137L1, CD137L5, and CD137L6 are respectively from amino acids 46-254, 83-254, 45-85, and 167-254 of the full-length amino acid sequence of CD137L ( figure 2 ). The nucleotide sequence of the protein is the sequence shown in SEQ ID NO.1 to SEQ ID NO.18, the amino acid sequence encoded by translation is the sequence shown in SEQ ID NO.19 to SEQ ID NO.36, and the connecting peptide is selected from SEQ ID NO. 56 to one of the sequences shown in SEQ ID NO.63.

[0073] The nucleus encoding the amino acid sequence of CD137L extracellul...

Embodiment 2

[0135] Example 2: Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in Bacillus subtilis expression system

[0136] 1. Construction of expression system

[0137] Using the pBluescriptII SK(+) vector constructed in Example 1 containing the complete recombinant protein gene sequence (SEQ ID NO.1-18) as a template, by designing different primers (5'PstI, 3'HindIII) and performing PCR, Amplify the different fusion protein genes connected to the pBluescriptII SK(+) vector and connect the complete fragment to the expression vector pP43 (the vector needs to be digested with PstI and HindIII before the ligation experiment). The operation steps such as PCR and enzyme digestion and connection are the same as the construction part of the fusion protein gene in Example 1. After the expression vector was constructed, the electroporation operation was carried out (for details, please refer to the instructions of the Bio-Rad electroporation instr...

Embodiment 3

[0142] Example 3: Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in yeast expression system

[0143] 1. Construction of expression system

[0144] The different fusion protein genes (SEQ ID NO.1-18) integrated in the pBluescriptII SK(+) vector were digested with EcoRI and NotI and ligated into pPIC9K to complete the vector construction. Subsequently, the electroporation operation was carried out (see the instruction manual of the Bio-Rad electroporation instrument for details), and the construction of the recombinant strain of Pichia pastoris GS115 was completed.

[0145] Expression of recombinant protein

[0146] Using BMGY enrichment bacteria, BMMY induced expression strategy to ferment to obtain the target product. On the first day, the expression strain constructed according to the above method was inoculated into the YPD seed medium, and cultivated overnight at 37°C. The next day, transfer an appropriate amount of seed cu...

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Abstract

The invention relates to fusion protein with double functions of inhibiting revascularization of a tumor microenvironment and activating adaptive immune response, and a gene and application thereof. The invention discloses a group of recombinant protein with Tumstatin and CD137L double functions, and is characterized in that the protein is formed by fusing Tumstatin active segment amino acid sequence (one of SEQ ID NO. 52 to SEQ ID NO. 55) and CD137L extracellular domain protein segment amino acid sequence (one of SEQ ID NO. 64 to SEQ ID NO. 66) through flexible connecting peptide. The protein and the protein coding gene can be applied to research and application of tumor angiogenesis inhibitors, various oncology related diseases and body immunity adjusting medicines.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. The invention relates to a fusion protein CD137L-Tumstatin and its gene, which has dual functions of inhibiting tumor microenvironment angiogenesis and activating adaptive immune response, as well as an expression vector containing the gene and a bacterial strain transformed by the vector, and also relates to the fusion The protein preparation method also involves the application of the protein with dual functions of CD137L and Tumstatin in the preparation of drugs for inhibiting tumor microvascular regeneration and related oncological diseases (such as melanoma, rectal cancer, lung cancer, etc.) and activating adaptive immune response, In addition, it involves the application of administration route (eg, oral administration, spray administration route, etc.). Background technique [0002] Carcinogenesis is a process in which multiple genes cooperate and different signaling pathways parti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/17A61K47/48A61P35/00A61P37/02
Inventor 刘楠马超何东洋陈依军
Owner CHINA PHARM UNIV
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