Preparation method of anti-human CD70 nano antibody for blocking combination of CD70 and ligand CD27 thereof and coding sequence of anti-human CD70 nano antibody
A nanobody and sequence technology, applied in the field of medicine and biology, to achieve the effects of small molecular weight, large storage capacity, shortening time and cost
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Embodiment example 1
[0053] Example 1: Construction of a single domain heavy chain antibody immune library targeting human CD70:
[0054] (1) Xinjiang Bactrian camels were immunized with eukaryotic human CD70 protein once every two weeks for a total of six consecutive immunizations. (2) After the six immunizations, the serum titer was detected by indirect ELISA. Lymphocytes in camel peripheral blood were isolated and total RNA was extracted (TRIZOL method). (3) According to TAKARA's Primescript Tm RT reagent kit with gDNA Eraser reagent instructions will extract the total RNA, such as figure 1 shown. Then reverse transcribe the RNA into cDNA, and then use the PCR method to amplify the VHH chain, including two rounds of PCR, the first round of PCR:
[0055] Upstream primer CALL01-Leader: 5'-GTCCTGGCTGCTCTTCTACAAGG-3'
[0056] Downstream primer CALL02-CH2: 5'-GGTACGTGCTGTTGAACTGTTCC-3'
[0057] The first round of PCR reaction conditions: pre-denaturation at 95°C for 5min, 95°C for 10s, 56°C fo...
Embodiment example 2
[0064] Example 2: Screening of anti-CD70 nanobody, identification and sequencing of positive clones
[0065] 1. Large-capacity CD70 phage nanobody library amplification
[0066] Inoculate the library in 100 mL of SB liquid medium (containing 10% glucose, 100 μg / mL ampicillin) to OD600 of 0.1, culture at 37 °C and 200 r / min until OD600 reaches 0.5, and add helper phage according to the multiplicity of infection 20:1 M13K07, 37°C, after static infection for 30 minutes, culture at 37°C, 100r / min for 30 minutes. Centrifuge the culture, resuspend the pellet with 50 mL of SB (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin), cultivate overnight at 30°C, 220 r / min, and centrifuge at 4°C, 8000rpm Add 5×PEG / NaCl solution and place on ice for 2h, centrifuge at 12000rpm for 30min, resuspend the pellet in phosphate buffer (PBS, 0.01 M, pH 7.4), and obtain the anti-CD70 single domain heavy chain antibody immune library , Take 10 μL to measure the titer of the amplified library.
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Embodiment example 3
[0096] Example 3: Expression and purification of CD70 nanobody
[0097] 1. Expression of CD70 nanobody
[0098] In this patent, the two positive clones identified by sequencing were not subcloned into other expression vectors, but the original vector pMECS and the original host strain TG1 were used for protein expression, and the clone was named TG1-pMECS-VHH. Streak the original strain on the SB-AG solid medium plate, put it upright for 10 minutes, and culture it upside down overnight. On the second day, single clones on the solid plate were picked and placed in 10 mL of SB-AG liquid medium containing 100 ug / mL ampicillin antibiotic, and cultured with shaking at 220 rpm at 37°C for 8 hours as bacterial seed liquid.
[0099] Inoculate the bacterial seed solution at 1% into SB-A liquid medium, culture at 37°C with shaking at 220rpm to OD600=0.8, add IPTG to a final concentration of 1mM, and cultivate overnight at 30°C at 150rpm. The next day, the supernatant was discarded by ...
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