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A kind of method and its application of producing antivascular agent rt-x in escherichia coli

A technology of Escherichia coli and RT-X, applied in the field of fusion protein RT-X technology and separation and purification, can solve the problem of RGD without literature reports

Active Publication Date: 2018-03-30
南通大学技术转移中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no literature report on modifying the sequence of Maspin protein to improve its targeting and anti-tumor activity in vivo, and there is no literature report on the synergistic inhibition of RGD and Maspin protein Angiogenesis

Method used

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  • A kind of method and its application of producing antivascular agent rt-x in escherichia coli
  • A kind of method and its application of producing antivascular agent rt-x in escherichia coli
  • A kind of method and its application of producing antivascular agent rt-x in escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083]Example 1 Cloning, expression, separation and purification of the vascular inhibitor RT-X gene

[0084] The Maspin gene refers to the cDNA sequence of Maspin (Genbank sequence number: AY893387.1), and the gene sequence is shown in SEQ ID NO:5;

[0085] ATGGATGCCCTGCAACTAGCAAATTCGGCTTTTGCCGTTGATCTGTTCAAACAACTATGTGAAAAGGAGCCACTGGGCAATGTCCTCTTCTCTCCAATCTGTCTCTCCACCTCTCTGTCACTTGCTCAAGTGGGTGCTAAAGGTGACACTGCAAATGAAATTGGACAGGTTCTTCATTTTGAAAATGTCAAAGATGTACCCTTTGGATTTCAAACAGTAACATCGGATGTAAACAAACTTAGTTCCTTTTACTCACTGAAACTAATCAAGCGGCTCTACGTAGACAAATCTCTGAATCTTTCTACAGAGTTCATCAGCTCTACGAAGAGACCCTATGCAAAGGAATTGGAAACTGTTGACTTCAAAGATAAATTGGAAGAAACGAAAGGTCAGATCAACAACTCAATTAAGGATCTCACAGATGGCCACTTTGAGAACATTTTAGCTGACAACAGTGTGAACGACCAGACCAAAATCCTTGTGGTTAATGCTGCCTACTTTGTTGGCAAGTGGATGAAGAAATTTCCTGAATCAGAAACAAAAGAATGTCCTTTCAGAGTCAACAAGACAGACACCAAACCAGTGCAGATGATGAACATGGAGGCCACGTTCTGTATGGGAAACATTGACAGTATCAATTGTAAGATCATAGAGCTTCCTTTTCAAAATAAGCATCTCAGCATGTTCATCCTACTACCCAAGGATGTGGAGGATGAGTCCACAGGCTTGGAGAA...

Embodiment 2

[0105] Example 2 Analysis of biological activity of fusion protein RT-X

[0106] 1. Endothelial Cell Migration Assay

[0107] Matrigel preparation: place the matrigel frozen at -80°C at 4°C overnight to become liquid; take 150 μL of Matrigel, coat the upper chamber of the Transwell (operated on ice), put it in a 37°C incubator, and incubate for 1 hour; digest HUVEC Cells were washed 3 times in serum-free medium, counted, and made into 2×10 6 Cell suspension; wash Matrigel-coated Transwell once with serum-free medium; add 100 μL cell suspension to each well; add 600 μL conditioned medium containing 20% ​​FBS to the lower chamber of Transwell; incubate for 48 hours in a 37°C incubator; take out Wash 2 times with PBS, wipe off the cells on the upper surface with cotton balls, fix with neutral formaldehyde for 10 minutes; add crystal violet (0.1%) to stain for 10 minutes, wash 2 times with PBS at room temperature, observe and take pictures under a microscope. Count the number of...

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Abstract

The present invention relates to the field of bioengineering and medical technology, in particular to a process and a separation and purification method for producing a soluble, biologically active, targeted anti-tumor angioinhibitory fusion protein RT-X in Escherichia coli, said fusion protein RT‑X contains RGD and Maspin proteins. The biologically active anti-vascular agent RT-X expressed and purified by the present invention can specifically and dose-dependently inhibit the migration of endothelial cells, and has the effect of inhibiting neovascularization in chicken embryo allantoic membrane animal experiments. The invention is applicable to the bioengineering pharmaceutical industry and the research of genetic engineering, biochemistry, molecular biology and the like.

Description

Technical field: [0001] The invention relates to the technical fields of bioengineering and medicine, in particular to a process and a separation and purification method for producing a soluble and biologically active fusion protein RT-X with targeting anti-tumor blood vessel inhibition in Escherichia coli. Background technique: [0002] In recent years, with the in-depth study of tumor anatomy and molecular biology, people have gradually realized that angiogenesis plays a key role in the growth and metastasis of solid tumors. Since Algureza proposed the concept of tumor-induced angiogenesis in 1947, until 1971, folkman clearly proposed the theory that tumor growth and metastasis depend on new angiogenesis. New blood vessels not only provide the necessary nutrients and oxygen for tumor growth, but also help eliminate metabolites , or the way of its distant metastasis (Folkman, J. What is the evidence that tumors are angiogenesis dependent? J. Natl. Cancer Inst. 1990: 82: 4-6...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12N15/62C07K19/00C07K1/36C07K1/34C07K1/22A61K38/57A61K47/42A61P35/00
Inventor 殷润婷张志强张伟
Owner 南通大学技术转移中心有限公司
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