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Recombinant protein Tumstatin-CD137L4 with Tumstatin activities as well as preparation method and application thereof

A technology of recombinant protein and activity, which is applied in the direction of recombinant DNA technology, medical preparations containing active ingredients, peptide/protein components, etc., can solve the problems of no research, etc., and achieve preparation avoidance, simple preparation, good application prospects and market value Effect

Inactive Publication Date: 2014-12-03
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been studies using vaccines of both properties to achieve this effect, no studies have fused CD137L and tumstatin to make a bifunctional, dual-target protein

Method used

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  • Recombinant protein Tumstatin-CD137L4 with Tumstatin activities as well as preparation method and application thereof
  • Recombinant protein Tumstatin-CD137L4 with Tumstatin activities as well as preparation method and application thereof
  • Recombinant protein Tumstatin-CD137L4 with Tumstatin activities as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Expression and purification of recombinant protein Tumstatin-CD137L4 with Tumstatin activity in yeast expression system

[0067] (1) Construction of recombinant protein Tumstatin-CD137L4 yeast expression vector with Tumstatin activity

[0068] The present invention designs 6 kinds of recombinant protein Tumstatin-CD137L4 expression vectors with Tumstatin activity, its nucleotide sequence is the sequence shown in SEQ ID NO.1 to SEQ ID NO.6, and the amino acid sequence is SEQ ID NO.7 to SEQ ID The sequence shown in NO.12. The histone or polypeptide consists of three groups of tumstatins with different active sites (three groups have amino acid sequences of 45-98, 60-132, and 60-98 positions of full-length tumstatin) and CD137L (with full-length CD137L extracellular domain protein) 50-240 amino acid sequence) are respectively connected and fused by Linker connecting peptides. Wherein the linker amino acid sequence is: GGGGSGGGGSGGGGS or AEAAAKEAAAKEAAAAKEAAAKA...

Embodiment 2

[0088] Example 2: Expression and purification of recombinant protein Tumstatin-CD137L4 with Tumstatin activity in Bacillus subtilis

[0089] (1) Construction of recombinant protein Tumstatin-CD137L4 Bacillus subtilis expression vector with Tumstatin activity

[0090] The present invention designs 6 kinds of recombinant protein Tumstatin-CD137L4 expression vectors with Tumstatin activity, its nucleotide sequence is the sequence shown in SEQ ID NO.1 to SEQ ID NO.6, and the amino acid sequence is SEQ ID NO.7 to SEQ ID NO The sequence shown in .12. The histone or polypeptide consists of three groups of tumstatins with different active sites (three groups have amino acid sequences of 45-98, 60-132, and 60-98 positions of full-length tumstatin) and CD137L (with full-length CD137L extracellular domain protein) 50-240 amino acid sequence) are respectively connected and fused by Linker connecting peptides. Wherein the linker amino acid sequence is: GGGGSGGGGSGGGGS or AEAAAKEAAAKEAAAA...

Embodiment 3

[0113] Example 3: Expression of recombinant protein Tumstatin-CD137L4 with Tumstatin activity in Escherichia coli expression vector and inclusion body protein renaturation and purification

[0114] (1) Construction of recombinant protein Tumstatin-CD137L4 with Tumstatin activity in Escherichia coli expression vector

[0115] The present invention designs 6 kinds of recombinant protein Tumstatin-CD137L4 expression vectors with Tumstatin activity, its nucleotide sequence is the sequence shown in SEQ ID NO.1 to SEQ ID NO.6, and the amino acid sequence is SEQ ID NO.7 to SEQ ID The sequence shown in NO.12. The histone or polypeptide consists of three groups of tumstatins with different active sites (three groups have amino acid sequences of 45-98, 60-132, and 60-98 positions of full-length tumstatin) and CD137L (with full-length CD137L extracellular domain protein) 50-240 amino acid sequence) are respectively connected and fused by Linker connecting peptides. Wherein the linker a...

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Abstract

The invention discloses a recombinant protein Tumstatin-CD137L4 with Tumstatin activities as well as a preparation method, related genes and application thereof. The recombinant protein has a tumstatin active fragment amino acid sequence and a CD137L extracellular region amino acid sequence, wherein the tumstatin active fragment amino acid sequence is selected from one of amino acid sequences shown as SEQ ID NO.13-SEQ ID NO.15 and the CD137L extracellular region amino acid sequence is shown as SEQ ID NO.16. The recombinant protein has the functions of inhibiting the proliferation of human umbilical vein endothelial cells and synergistically stimulating the proliferation of T cells and can be used for preparing angiogenesis inhibitors and drugs for various tumor related diseases, retinopathy, cell proliferation, organism cytokines synthesis and secretion and organic immunity adjustment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a group of recombinant protein Tumstatin-CD137L4 with Tumstatin activity and its gene, an expression vector containing the gene and a bacterial strain transformed by the vector, a preparation method of the protein, and a The protein and its gene play an important role in the preparation of angiogenesis inhibitors, various tumor-related diseases (such as melanoma, prostate cancer, lung cancer, colorectal cancer, kidney cancer, bladder cancer, etc.), retinopathy, cell proliferation, body cytokine synthesis and secretion , Regulating body immunity and other drugs, and the application of the recombinant protein or gene in the preparation of related dosage forms such as oral or injection drugs. Background technique [0002] A large number of studies have proved that poor immunogenicity and abundant angiogenesis are two major characteristics of tumor tissue, and they ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/17A61P35/00A61P37/02A61P27/02A61P9/00
Inventor 王淑珍高振月何东洋陈依军
Owner CHINA PHARM UNIV