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Recombination glant associated virus carrier induced TNF concerned cytonecrosis ligand peptide segment and its use

A tumor cell and vector technology, which can be used in tumor necrosis factor, cells modified by the introduction of foreign genetic material, anti-tumor drugs, etc. Effects of growth and formation inhibition

Inactive Publication Date: 2004-12-08
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of TRAIL (114-281) recombinant expression vector in its technology is complicated, and it has not been reported whether it can be specifically expressed in the liver and enter the blood circulation, nor whether it can be used for gene therapy of tumors such as liver metastases and lung cancer

Method used

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  • Recombination glant associated virus carrier induced TNF concerned cytonecrosis ligand peptide segment and its use
  • Recombination glant associated virus carrier induced TNF concerned cytonecrosis ligand peptide segment and its use
  • Recombination glant associated virus carrier induced TNF concerned cytonecrosis ligand peptide segment and its use

Examples

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experiment example 1

[0035] Using TRAIL full-length cDNA cloned from Chinese normal human peripheral blood lymphocytes as a template, synthetic TRAIL 95-281 The cDNA upstream and downstream oligonucleotide sequences are used as primers to establish a PCR amplification reaction. TRAIL 95-281 The nucleotide sequences of the cDNA upstream and downstream oligonucleotide primers are 5’-TA respectively GAATTC ACCATGACCTCTGAGGAAACCATT-3’ and 5’-CCC AAGCTT TTAGCCAACTAAAAAGGCCC-3’. Introduce the Kozak sequence into the primers and add restriction enzymes EcoRI and HindIII restriction sites at both ends. The PCR amplification conditions were 94°C for 45sec, 58°C for 1min, 72°C for 1min, 30 cycles, and finally extension at 72°C for 15min. After the PCR product was digested with EcoRI and HindIII, it was inserted into the pAM / CAG / EGR-1-pL-WPRE-BGHpolyA AAV expression vector that had been digested with EcoRI and HindIII restriction enzymes in advance. Recombinant TRAIL 95-281 -AAV expression plasmid and helper vi...

experiment example 2

[0037] See Figure 1A, Western Blot: Liver tissue or cells are lysed in a lysis solution (1×PBS containing 1% Nonidet P-40, 0.35mg / ml PMSF, 9.5μg / ml leupeptin and 13.7μg / ml pepstatinA) and centrifuged at 12000rpm After removing the precipitation, the supernatant was subjected to SDS-PAGE gel electrophoresis after the protein concentration was determined by the BCA protein analysis kit (Pierce Chemical Company, Rockford, IL), transferred to the PVDF membrane and blocked at 4°C overnight, and the primary antibody (anti-TRAIL After cloning antibody, diluted 1:1000 in blocking solution, incubate for 2h at room temperature, add secondary antibody (HRP-labeled rabbit anti-goat antibody diluted in blocking solution 1:3000), incubate at room temperature for 1h, wash thoroughly, and then use ECL System (Amersham Pharmacia Biotech Co.) for protein color development. Check TRAIL 95-281 For the expression in mouse serum, the serum was collected from the tail vein of the mouse, concentrated 10-...

experiment example 3

[0039] See Figure 1B-D, TRAIL 95-281 Expression and biological activity determination of HEK-293 cells in Dulbecco's modified essential medium (DMEM) complete medium (that is, in DMEM medium with 10% fetal bovine serum, 100U / ml penicillin and 100μg / ml streptomycin Cultured in) for more than 6 hours, washed with 1×PBS and added recombinant virus of different titers (medium is DMEM with 2% fetal bovine serum), and added DMEM complete medium after 8 hours. The HEK-293 cell culture medium transduced by the recombinant virus was collected at different time points, and the culture medium was frozen and concentrated 10 to 20 times in a vacuum to determine the protein concentration and perform an ELISA test. Approximately 100μg of protein in 0.05M Na 2 CO 3 (pH 9.6) After dilution, a 96-well plate was coated at 37°C. After coating for 2h, it was blocked with blocking solution (1×PBS with 0.05% Tween-20 and 5% skimmed milk powder) at 37°C for 0.5h. Add anti-TRAIL polyclonal antibody (dilut...

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Abstract

The No 95-281 peptide fragments of cell wither ligand (TRAIL) associated with recombinant tumor necrosis factor carried by adeno-associated virus carrier, its active form for expressing tripolymer in body, and its application in preparing medicines for treating transferred liver tumor and lung cancer are disclosed.

Description

Technical field [0001] The invention relates to a recombinant tumor necrosis factor-related apoptosis inducing ligand (TRAIL) peptide fragment carried by an adeno-associated virus (AAV) expression vector and a preparation method thereof , Packaging, in vivo expression and its use, especially the recombinant expression vector (TRAIL) which encodes the 187 amino acid residues from 95 to 281 of TRAIL and the adeno-associated virus 95-281 -AAV) and its use. The recombinant virus of the present invention can be used alone or in combination with other anti-cancer drugs for gene therapy of tumors such as liver metastases and lung cancer. technical background [0002] TRAIL (tumor necrosis factor-related apoptosis inducing ligand) belongs to the tumor necrosis factor (TNF) family member, which was cloned and named by Wiley in 1995. Studies have found that TRAIL can rapidly induce apoptosis in a variety of human tumor cells such as breast cancer, rectal cancer, lung cancer and prostate ca...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61K48/00A61P35/00C07K14/525C12N5/10C12N15/28C12N15/33C12N15/63
Inventor 郑德先许瑞安马宏刘彦信孔祥复
Owner OBIO TECH SHANGHAI CORP LTD
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