Tumor-lysing virus growing selectively in tumor cells
An adenovirus and gene technology, applied in the field of oncolytic virus selectively proliferating in tumor cells, can solve the problems of low efficiency of gene introduction, unsatisfactory therapeutic effect, and inability to detect telomerase activity, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0064]
[0065] Using specific primers (E1A-S: sequence 5, E1A-AS: sequence 6), RT-PCR was performed on the RNA extracted from 293 cells to amplify the 899bp E1A gene. The DNA extracted from 293 cells was subjected to DNA-PCR using primers (E1B-S: SEQ ID NO: 7, E1B-AS: SEQ ID NO: 8) to amplify the 1823 bp E1B gene.
[0066] The respective PCR products were subjected to TA cloning (TA cloning kit double promoter; Invitrogen), and after the sequence was confirmed, DNA fragments of 899 bp (E1A) and 1823 bp (E1B) were cut out by restriction enzyme EcoRI, respectively.
[0067] E1A and E1B (E1A-IRES-E1B) were respectively inserted in the forward direction at the M1uI cleavage site and the SalI site of the pIRES vector (CLONTECH).
[0068] The 455 bp hTERT promoter sequence cleaved with restriction enzymes M1uI and Bg1II was inserted forward into the XhoI site upstream of E1A of E1A-IRES-E1B (phTERT-E1A-IRES-E1B).
[0069] The cytomegalovirus (CMV) promoter contained in the pShut...
Embodiment 2
[0072]
[0073] Immortalized human vascular endothelial cells HUVEC, human normal fibroblasts ( In 10 kinds of cells of W138, NHLF), use RNAzol (Cinna / Biotecx) to extract RNA, use LightCycler and LightCycler DNA TeloTAGGG Kit (Roche Molecular Biochemicals), perform real-time quantitative reverse transcription (RT)-PCR, and compare the hTERT of each cell gene expression level. The result is shown in Figure 2.
[0074] Comparing A549 cells with the highest expression level as 1.0, it was confirmed that hTERT gene expression was 0.18 to 1.00 in A549, H226Br, H1299, SW620, DLD-1, LoVo and other cancer cells and 293 cells. However, in immortalized HuVEC cells Its expression was not detected in normal cells such as WI38 and NHLF.
Embodiment 3
[0076]
[0077] Human colorectal cancer cells SW620 and human normal fibroblasts WI38 were cultured in vitro, and RNA was recovered after 36 hours after infection with TRAD at concentrations of 0.1 and 1 MOI (multiplicity of infection). 293 cells were used as a positive control.
[0078] Reverse transcription (RT) was performed using the GeneAmp RNA PCR core kit, and the primers for the E1A and E1B genes were used for 30 cycles of amplification through the GeneAmp PCR system 9700 thermal cycler (PE Applied Biosystems). PCR products were electrophoresed on a 1.2% agarose gel and visualized by ethidium bromide staining (2 spots on Figure 3A). Band intensities were measured on a gel image analyzer and quantified and plotted using GAPDH as an internal standard (Fig. 3A bottom).
[0079] Human colorectal cancer cells SW620 and human normal fibroblasts WI38 were cultured in vitro, and the adsorbed cells were recovered after infecting TRAD at a concentration of 0.1 and 1 MOI for 4...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More