Conditional replicative adenoviral vector and application of viral replication regulated by transcriptional repressive tet-on system
A tet-on, virus replication technology, applied in the field of life sciences, can solve the problems of ineffective virus delivery, lysis, loss of vitality, etc., to achieve the effect of increasing the virus load
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Embodiment 1
[0030] Conditionally it is replicating adenoviral vectors according to the present embodiment constructed virus replication by transcription Tet-on system suppressor TetR-KRAB regulation mediated follows:
[0031] Adenovirus E1A-E1B19KD adenovirus E1 region comprises elements for the connection sequence 5'-CMV promoter, TetR-KRAB gene, tetracycline responsive promoter element TRE3G-E1b Pro, EcoRⅠ restriction sites, deletions of the protein E1BΔ55KD sequences, restriction sites SpeⅠ, mRNA transcription termination signals SV40polyA-3 '; CMV promoter expression transcriptional repressor TetR-KRAB; EcoRⅠ two restriction sites SpeI and inserted into the gene sequence for translation start site of the adenovirus E1A point adenovirus E1B 19KD protein gene stop codon fragment, containing the tetracycline responsive promoter element TRE3G-E1b Pro adenovirus expressing E1A gene, the adenovirus E1B 19KD gene by the expression of its own promoter, adenovirus obtained after packaging named Ad...
Embodiment 2
[0057] Conditionally replicating adenovirus viral replication by inhibiting the transcriptional regulation of type Tet-on system TetR-KRAB vector-mediated infection of human cervical carcinoma Hela cells in the regulation of replication
[0058] Same conditions replicative adenovirus vector virus replication according to the present embodiment constructed by the transcriptional repressor Tet-on system type TetR-KRAB regulation mediated described in Example 1.
[0059] Which steps are as follows:
[0060] Example 1-4 Step this embodiment is identical to Example 1.
[0061] Conditionally replicating adenovirus 5, inhibition of viral replication under the transcriptional regulation of type Tet-on system TetR-KRAB vector-mediated infection of Hela cells
[0062] Hela cells, after the mass fraction of 0.1% trypsin, by 5 × 10 4 / Well plated on 24-well plate, and 600μL was added NCS containing 10% DMEM culture overnight. Every other day of infection, the virus Ad5-CMV-TetR-KRAB-TRE3G-E1...
Embodiment 3
[0094] Replication regulation U87MG tumor cells after conditionally replicative adenovirus viral replication by inhibiting the transcriptional regulation of type Tet-on system TetR-KRAB vector-mediated infection of human malignant glioblastoma brain
[0095] Same conditions replicative adenovirus vector virus replication according to the present embodiment constructed by the transcriptional repressor Tet-on system type TetR-KRAB regulation mediated described in Example 1.
[0096] Which steps are as follows:
[0097] Example 1-4 Step this embodiment is identical to Example 1.
[0098] Cells used in method step 5-7 which is replaced by Hela cells in Example 2 of U87MG cells, remaining the same manner as in Example 2.
[0099] Viral genome replication see test results Figure 4The results show, conditionally replicating adenovirus embodiment virus present embodiment replication by TetR-KRAB mediated transcriptional suppression type Tet-on system regulated Ad5-CMV-TetR-KRAB-TRE3G-E1bP...
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