Application of immune regulation markers TIPE2 (Tumor Necrosis Factor-a Induced Protein 8 like 2) in preparation of medicine used for treating vascular proliferation diseases
A technique for vascular proliferation and immune regulation, applied in the field of recombinant adenovirus, to achieve the effect of inhibiting excessive proliferation and broad development and application prospects
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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, cDNA reverse-transcribed from the total RNA of C57BL / 6...
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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