Tumor suppressing polypeptide TSF and gene therapy vector composition
A technology of composition and polypeptide substance, applied in gene therapy, drug combination, anti-tumor drug, etc., can solve the problem of limited pharmacokinetics in the preparation process, and achieve the effect of eliminating interference
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Embodiment 1
[0008] Cloning of embodiment 1.TSF cDNA and packaging of recombinant adenovirus
[0009] Total RNA was extracted from normal human peripheral blood cells using TRIZOL kit (product of Life Technologies). TSF cDNA was amplified by RT-PCR, and the PCR reaction conditions were pre-denaturation at 94°C, 30”; annealing at 58°C, 45”; extension at 72°C, 2’, 30 cycles. The amplified product is 1.7Kb in length. The amplified product was detected by an automatic DNA sequencer, and the amplified product was completely consistent with the designed sequence. The PCR primers used were designed according to the full-length cDNA sequence of TSP1 (GENE BANK Accession Code NM003246), specifically: the upstream primer 5'-AAT CGA GGT ACC GTA CAC ACA GGA TCC CTG CTG-3'; the downstream primer 5'-GAT CCC AAG CTT TTA AAT TGG ACA GTC CTG CTT GTT-3'. The TSF cDNA coding region is 40-1683.
[0010] Digest the TSF cDNA and the shuttle plasmid pShuttle-CMV in the adenovirus packaging system (product of...
Embodiment 2
[0011] Example 2. RT-PCR and Western blot detection of TSF recombinant adenovirus infection activity
[0012] The human leukemia cell line K562 was infected with TSF recombinant adenovirus, control virus and PBS, and the MOI of infection was 100. After 48 hours, the cells were collected to extract total RNA with TRIZOL kit (product of Life Technologies Company), and the One step RT- PCR kit was used to detect the expression of TSF at the transcriptional level in K562 cells ( figure 2 ). The PCR reaction conditions and primers used are as described in Example 1. At the same time, the culture supernatant of K562 cells 48 hours after virus infection was collected to detect the expression of TSF protein level by Western blotting. Specifically, after the culture supernatant was concentrated, the total protein was separated by 9% SDS-polyacrylamide gel electrophoresis, and the latter was electrotransferred to a PVDF membrane. The antibody was incubated with PVDF membrane, and DA...
Embodiment 3
[0013] Example 3. Therapeutic Experiment of TSF Recombinant Adenovirus on K562 Nude Mice Transplanted Tumors
[0014] Female, 5-6 week old Balb / c nude mice were subcutaneously inoculated with 10 7 K562 cells were randomly divided into 3 groups when the tumor body diameter was about 5-7mm, 6 mice in each group, and 10 mice were injected into the transplanted tumor respectively. 9 pfuTSF recombinant adenovirus, 10 9 The control virus and PBS were used, and the above treatment was repeated on the 3rd and 10th day thereafter. Measure the tumor size of each group every three days from the beginning of treatment, according to the formula V=L×W 2 Calculate, where L is the long diameter of the tumor, and W is the short diameter. The nude mice were sacrificed 21 days after treatment, and the tumor mass was removed for histological examination. The results showed that TSF recombinant adenovirus could significantly inhibit the growth of K562 nude mouse xenograft tumor ( Figure 4 )....
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