TAT-PDCD4 (programmed cell death 4) fusion protein and application of TAT-PDCD4 fusion protein in drug for treating ovarian cancer
A fusion protein, the technology in the sequence listing, applied in the biological field to achieve the effects of inhibiting proliferation and migration, no toxic side effects, and high transmembrane rate
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Embodiment 1
[0040] Embodiment 1 Construction of TAT-PDCD4-His recombinant expression vector
[0041] Construction of the fusion gene: design primers according to the DNA sequence of SEQ ID No.5 (see Table 1 below), fuse the gene sequence by primer synthesis PCR method, and clone it into the expression vector pET30a(+).
[0042] Table 1 Primers
[0043]
[0044] 1. Obtain the cloned target sequence by PCR:
[0045] a. Full-length PCR: see Table 2 for the reaction system:
[0046] Table 2 One round reaction system of full-length PCR reaction system
[0047]
[0048] Reaction condition: 95°C for 3min
[0049]
[0050] b. Recover the PCR product of the above steps by glue: the above product is recovered by tapping the rubber.
[0051] c.Gibson recombination reaction experiment
[0052] 1) Simultaneously with PCR, the pET30a(+) plasmid was treated with NdeI / BamHI for gel recovery.
[0053] 2) Gibson recombination reaction system: Add 5 ul of the above-mentioned PCR recovery prod...
Embodiment 2
[0056] Induced expression and purification of embodiment 2 TAT-PDCD4-His fusion protein in Escherichia coli
[0057] Transform the recombinant cloning plasmid with correct sequencing into Escherichia coli BL21 (DE3) competent cells, pick a single clone colony and inoculate it into 2ml PBL medium (containing kanamycin) for overnight culture on a shaker. The next day, inoculate into large-volume LB medium at a ratio of 1:100, culture at 250 rpm until the OD 600 is between 0.3-0.5, then add an appropriate amount of inducer IPTG, continue to culture on a shaker for 6 hours, and centrifuge to discard the supernatant for later use. cassette for protein purification and endotoxin removal. Then carry out SDS-PAGE and Western-blot analysis to it, such as figure 1 , Judging from the molecular weight, the protein obtained in lane 1 is a TAT-PDCD4 fusion protein. Therefore, the TAT-PDCD4-His fusion protein was successfully expressed and purified in this experiment.
Embodiment 3
[0058] Example 3 Study on the transmembrane velocity of TAT-PDCD4-HIS
[0059] 1) Inoculation of cells Ovarian cancer cell-SKOV3 was digested with trypsin, counted, and then inoculated into 24-well cell culture plates with coverslips placed in advance. Incubate overnight at 37°C, 5% CO2.
[0060] 2) Fusion protein TAT-PDCD4 stimulates the cells to discard the supernatant, add TAT-PDCD4 protein containing 5ug / ml, and continue culturing for 30 minutes, 1 hour and 3 hours respectively.
[0061] 3) Fix with 4% paraformaldehyde for 10 minutes before staining antibody treatment (300ul per well), then incubate with mouse anti-human PDCD4 antibody for 1 hour, then treat with FITC-rabbit anti-mouse secondary antibody for 30 minutes, and then treat cells with DAPI 3 Minutes, finally take out the coverslip, place the cells on the slide with the cells facing down, add an appropriate amount of anti-fade agent, and seal the slide for later use.
[0062] 4) Laser confocal microscope observ...
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