High affinity soluble pdl-1 molecule
A PDL-1, PD-1 technology, applied in peptide/protein components, chemical instruments and methods, recombinant DNA technology, etc., can solve the problems of weakening anti-tumor immune response, tumor immune escape, etc.
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Embodiment 1
[0099] Expression, renaturation and purification of the wild-type PDL-1 of embodiment 1
[0100] The extracellular amino acid sequence and nucleotide sequence of wild-type PDL-1 are SEQ ID NO:1 and 2 respectively, such as Figure 1a and Figure 1bAs shown, the target gene carrying the extracellular sequence of wild-type PDL-1 was digested with NcoI and NotI, and then ligated with the pET28a vector that was digested with NcoI and NotI. The ligation product was transformed into E.coli DH5α, coated with kanamycin-containing LB plates, cultured upside down at 37°C overnight, and positive clones were picked for PCR screening, and the positive recombinants were sequenced, and the recombinant plasmid was extracted and transformed after confirming the correct sequence to E.coli BL21(DE3) for expression.
[0101] All the BL21(DE3) colonies containing the recombinant plasmid pET28a-PDL-1 were inoculated in LB medium containing kanamycin, cultured at 37°C until the OD600 was 0.6-0.8, an...
Embodiment 2
[0104] Example 2 Combination Characterization
[0105] BIAcore analysis
[0106] The BIAcore T200 real-time analysis system was used to detect the binding activity of wild-type PDL-1 molecules to PD-1. Add the anti-streptavidin antibody (GenScript) to the coupling buffer (10mM sodium acetate buffer, pH 4.77), and then flow the antibody through the CM5 chip activated with EDC and NHS to immobilize the antibody on the chip surface , and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process, and the coupling level was about 15,000 RU.
[0107] Let a low concentration of streptavidin flow over the surface of the antibody-coated chip, then flow biotinylated PD-1 through the detection channel, and the other channel as a reference channel, and then add 0.05mM biotin in 10μL / min flow through the chip for 2 minutes to block the remaining binding sites of streptavidin. The affinity was determined by a single...
Embodiment 3
[0114] Example 3 Production of High Affinity PDL-1 Molecules
[0115] Using the extracellular sequence of the wild-type PDL-1 described in Example 1 as a template strand, according to the phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354), high-affinity Screening for PDL-1. After several rounds of screening, the phage library had a strong binding signal to PD-1, and a single clone was picked from it for sequence analysis.
[0116] Express, refold and purify the high-affinity PDL-1 molecule of the present invention according to the method described in Example 1, and determine its affinity with PD-1 molecule according to the method described in Example 2. The affinity between the high-affinity PDL-1 molecule obtained in the present invention and the PD-1 molecule is at least 2 times that of the wild-type PDL-1 molecule and the PD-1 molecule, and its amino acid sequence is shown in Figure 5. The affinity value of 1 molecule is shown ...
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