Anti-H5N1 virus intracellular antibody PTD-3F-mFc and application of anti-H5N1 virus intracellular antibody PTD-3F-mFc
A technology of 1. ptd-3f-mfc, 2. ptd-3f-mfc, applied in the application field of preparing anti-H5N1 type human avian influenza virus drugs
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Embodiment 1
[0020] Example 1 Construction and expression of M1 recombinant expression plasmid pET-SUMO-M1 and purification of M1 protein
[0021] Design and synthesize specific primers P1 and P2 for the M1 protein, use the H5N1 virus cDNA as a template to PCR amplify the M1 protein gene, and clone it into the PET-SUMO vector to construct the plasmid pET-SUMO-M1, and then transfer it into the T-shot sensory Initial screening was carried out on the agar plate containing kanamycin resistance; a single colony was selected and cultured in LB liquid medium; the plasmid was extracted with a plasmid recovery kit and identified by PCR, and the product was analyzed by 1% agarose gel electrophoresis. A band of about 750 bp was obtained, the size of which was consistent with the inserted target gene, and the sequence was determined to prove that the target fragment was correctly inserted into the vector, and the recombinant plasmid pET-SUMO-M1 was successfully constructed.
[0022]The recombinant pla...
Embodiment 2
[0024] Example 2 Amplification of phage single-chain antibody library
[0025] Inject all the frozen Tomlinson I and J libraries into 200 mL 2×TY medium (containing 100 µg / mL Amp and 1% glucose), and culture with shaking at 37°C until the OD600 value is about 0.4, from Take out 50 mL bacterial liquid from the culture medium, add 2×10 11 The helper phage KM13 was placed in a water bath at 37°C for 30 minutes, centrifuged at 3000×g for 10 minutes at 4°C, and the pellet was resuspended with 50 mL of 2×TY medium (containing 100 µg / mL Amp, 50 µg / mL Kan and 0.1% glucose). Suspended and cultured overnight at 30°C with shaking. Centrifuge the overnight product at 4°C, 3500×g for 30 min, collect 40 mL of the supernatant, add 10 mL of ice-cold PEG / NaCl solution (final concentration is 20% PEG-6000, 2.5 mol / L NaCl), mix well and store on ice Place for more than 1 h, centrifuge at 4°C, 3500×g for 30 min, discard the PEG / NaCl solution, resuspend the pellet in 2 mL of PBS, centrifuge at 1...
Embodiment 3
[0026] Example 3 Screening of anti-M1-scFv
[0027] Coat the purified M1 protein on a 96-well microtiter plate overnight at 4°C. Discard the supernatant the next day, block with 2% Milk-PBS at 37°C for 2 hours, add the prepared secondary phage antibody library, incubate with vigorous shaking at room temperature for 60 minutes, discard the liquid after standing for 60 minutes, and replace with PBS containing 0.1% Tween-20 Wash 10 times, after washing, gently pat dry the remaining liquid in each well, add 50 µL eluent (5 mg / mL trypsin-PBS) to each well, shake vigorously at room temperature for 10 min, elute phage, collect and store at 4 °C .
[0028] Infect E.coli TG1 with the eluted phage, spread on TYE plates (containing 100 μg / mL ampicillin and 1% glucose) and culture overnight at 37°C. The phage library was amplified using the helper phage KM13, and the phage were recovered by PEG / NaCl. Repeat the above process 3 times, a total of 4 rounds of screening.
[0029] Phage in...
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