Screening method of vGPCR protein targeting peptide, chimeric toxin and application thereof
A screening method and protein target technology, applied in the field of vGPCR protein targeting peptide screening, can solve problems such as poor prognosis and no optimal treatment plan
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Embodiment 1
[0027] Example 1: Screening of short peptides specifically binding to vGPCR proteins
[0028] The specific operation is as follows:
[0029] 1. Lentiviral packaging and stable cell line construction
[0030] 1. Plasmid construction: vGPCR and GFP were inserted into the pLVX IRES Puro vector with EcoRI / XbaI and XbaI / NotI. After successful sequencing, STBL3 was used to amplify, and VSVG and pSPAX were simultaneously amplified and plasmids were extracted.
[0031] 2. Cell plating: Inoculate HEK293T on a 10cm dish, and grow the cell density to 60-70%.
[0032] 3. Lentiviral packaging:
[0033] 1) Measure 1.25 μg of VSVG, 3.5 μg of pSPAX2 and 5 μg of recombinant plasmid into 500 μL of blood-free and anti-antibody-free DMEM medium, add 30 μL of PEI, mix thoroughly and let stand for 20 minutes.
[0034] 2) After 6 hours of transfection, replace with complete medium, and collect the supernatant at 24, 48, and 72 hours.
[0035] 4. Lentivirus purification: centrifuge at 4000rpm for...
Embodiment 2
[0084] Example 2: Using immunofluorescence to verify the affinity of vGPCR proteins and candidate short peptides
[0085] The specific operation is as follows:
[0086] 1. Short peptide synthesis: Coupling the N-terminal of the candidate polypeptide fragments corresponding to the three positive clones (1C3, 1F10, 1B5) with strong vGPCR binding ability in the figure to biotin (1C3: Biotin-LR; 1F10: Biotin- FL; 1B5: Biotin-SR), this part was completed by Shanghai Gill Biological Co., Ltd.
[0087] 2. Cell plating: Put the cell slides into a 6-well plate, wash with ice PBS three times, seed GFP / HEK293T and vGPCR-GFP / HEK293T cells in a 6-well plate overnight, and the cell density reaches 60-70%;
[0088] 3. Fix the cells: Aspirate the culture medium, wash twice with ice-cold PBS, fix with 4% paraformaldehyde at room temperature for 30 min.
[0089] 4. Blocking: After washing with PBS three times, add 0.2% FSG blocking solution, and block at room temperature for 0.5 h.
[0090] ...
Embodiment 3
[0095] Example 3: Specific killing activity of chimeric toxin FL-Lytic on KSHV-related tumor cells
[0096] The specific operation is as follows:
[0097] 1. Short peptide synthesis: Synthesis of chimeric toxin (FL-Lytic) and cleavage peptide (Lytic) as a negative control, this part was completed by Shanghai Qiangyao Biotechnology Co., Ltd.
[0098] FL-Lytic:FGDRTITQMSQLGGGKLLLKLLKKLLKLLKKK
[0099] Lytic: KLLLKLLKKLLKLLKKK
[0100] 2. Cell killing activity test: 25,000 KSHV-related tumor cells (BCBL-1, BCP-1 are KSHV-positive cells, BJAB and DG75 are KSHV-negative cells) were planted in 96-well plates (25,000 / 50 μL containing 10% FBS 1640 medium), then add 50 μL of different concentrations of chimeric toxin solutions (0, 2.5, 5, 10 μmol / L) prepared in 1640 medium with 10% FBS, and treat at 37 ° C for 2 h under the condition of 5%. Cell viability was detected by MTT reagent.
[0101] The result is as Figure 4 As shown, it was found that for KSHV-positive cells, the chime...
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