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Application of immunomodulatory molecule tipe2 in the preparation of drugs for the treatment of angioproliferative diseases

A purpose, primer design technology, applied in the field of recombinant adenovirus, to achieve broad development and application prospects, and the effect of inhibiting excessive proliferation

Active Publication Date: 2015-10-28
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It has been confirmed by authoritative organization that the use of TIPE2 recombinant adenovirus to inhibit the proliferation of vascular VSMCs, thereby preventing the formation of postoperative vascular restenosis, has not been reported at home and abroad.

Method used

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  • Application of immunomodulatory molecule tipe2 in the preparation of drugs for the treatment of angioproliferative diseases
  • Application of immunomodulatory molecule tipe2 in the preparation of drugs for the treatment of angioproliferative diseases
  • Application of immunomodulatory molecule tipe2 in the preparation of drugs for the treatment of angioproliferative diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of mouse TIPE2 gene pRK5 expression vector

[0040] (1) Acquisition of mouse TIPE2 gene CDS region

[0041] PCR primer design:

[0042] According to the principles of primer design, primers were designed to amplify the CDS region of the mouse TIPE2 gene, and restriction sites for BamH1 and Xho1 were added to the upstream and downstream of the primers respectively to facilitate subsequent plasmid construction. In addition, a Kozak's sequence that facilitates transcription is added between the upstream primer restriction site and the initiation codon ATG. The specific primer sequences are as follows:

[0043] mTIPE2BamH1F:5'-ATTATTGGATCCGCCACCATGGAGTCCTTCAGCTCAAAGA-3';

[0044] mTIPE2Xho1R: 5'-ATTATTCTCGAGTCAGAGCTTGCCCTCGTCCAGC-3'; as shown in sequences 3 and 4 in the sequence listing.

[0045] (2) Sequence amplification of mouse TIPE2 gene CDS region

[0046] Using 2×Taq PCR Mix products, the cDNA reverse-transcribed from the total RNA of C57...

Embodiment 2

[0061]Example 2: Construction of mouse TIPE2 adenovirus expression vector and virus packaging

[0062] Construction of the pShuttle-GFP-CMV-TIPE2 shuttle plasmid: BamH I and Xho I double-digested the pRK5-TIPE2 plasmid (preserved in this unit, see Example 1 for the construction method) and the pShuttle-GFP-CMV shuttle plasmid, recovered the TIPE2 target gene fragment and Vector, enzyme-linked to obtain pShuttle-GFP-CMV-TIPE2 plasmid.

[0063] Construction of pADxsi-GFP-CMV-TIPE2 virus plasmid: use restriction endonuclease I-CeuⅠ+I-SceⅠ to double digest the pADxsi backbone plasmid and pShuttle-GFP-CMV-TIPE2 plasmid, link the target fragment with pADxsi enzyme, and connect the product Transform Escherichia coli DH5α to carry out homologous recombination in the bacteria. After screening positive clones on antibiotic plates, they were amplified in large numbers and named as pADxsi-GFP-CMV-TIPE2 viral plasmid.

[0064] Virus packaging: linearize the recombinant adenovirus plasmid ...

Embodiment 3

[0067] Example 3: Establishment of mouse carotid artery injury model

[0068] (1) Mice were anesthetized and fixed: 8-10 weeks old SPF grade C57BL / 6J mice were weighed, and 0.8% sodium pentobarbital was injected intraperitoneally at 1 mL / 200 g body weight. After the mice were deeply anesthetized, the limbs and head of the mice were fixed in the supine position on the mouse operating table.

[0069] (2) A midsagittal incision was made in the neck after povidone iodine disinfection, and the submandibular gland was carefully separated to expose the left common carotid artery.

[0070] (3) The left common carotid artery was separated, and the vagus nerve attached to it was carefully separated with a glass needle, and then the carotid artery was completely ligated with surgical thread.

[0071] (4) Restore the submandibular gland, suture the incision, and take care of the mouse until it wakes up.

[0072] (5) After the operation, the mice were kept in an SPF environment for the f...

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Abstract

The invention discloses an application of immune regulation markers TIPE2 (Tumor Necrosis Factor-a Induced Protein 8 like 2) in preparation of a medicine used for treating vascular proliferation diseases, and also discloses a recombinant adenovirus for expressing the TIPE2. The recombinant adenovirus for the TIPE2 is adopted to realize the in-vivo continuous high expression of TIPE2 protein, which shows the function on inhibiting excess proliferation of a vascular smooth muscle cell, inhibits the hyperplasia of neointima caused by the vascular injury in order to reduce vascular stenting postoperative complication and prognosis and other postoperative complications and prognoses, creates a new path with a wide prospect on effectively preventing and treating the vascular in-stent restenosis of acute coronary syndrome and intervening other vascular proliferation diseases on the cytological basis of vascular smooth muscle proliferation in clinic, and also provides the reference for developing a novel medicine.

Description

technical field [0001] The invention relates to a recombinant adenovirus highly expressing TIPE2 protein and its application, in particular to the application of TIPE2 as a new drug for preventing and treating vascular proliferative diseases such as restenosis after vascular surgery. Background technique [0002] Coronary artery disease has always been one of the diseases with high morbidity and high mortality in developed countries. With the development of economy, it has gradually spread to developing countries. Today, the treatment of coronary artery disease mainly adopts percutaneous coronary intervention (PCI). Most PCI treatments involve the placement of coronary stents. Coronary artery stent therapy has a success rate of up to 90% and high reproducibility. It has developed rapidly since its clinical application, and has become the main means of myocardial revascularization [see: Antonio Curcio, Daniele Torella, Ciro Indolfi.Mechanisms of Smooth Muscle Cell Prolifera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17C12N7/01A61K48/00A61P9/00A61P35/00A61P27/02
Inventor 刘素侠章桂忠
Owner SHANDONG UNIV
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