Nano antibody for hypoxia inducible factor to 1 alpha and coding sequence thereof
A hypoxia-inducible factor and nano-antibody technology, applied in genetic engineering, plant genetic improvement, anti-animal/human immunoglobulin, etc.
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Embodiment 1
[0022] The preparation process of the HIF-1α-PAS-B domain protein, wherein the HIF-1α-PAS-B domain protein is a recombinant protein with a GST tag:
[0023] (1) The primers were designed according to the coding gene sequence of the HIF-1α-PAS-B domain in the NCBI database (NCBI ReferenceSequence: NM_001530.3),
[0024] Upstream primer HIF-1α-PAS-B+:5’-CGC GGATCC ATTCCTTTAGATAGCAAGACTTTC-3' (SEQ ID NO: 3)
[0025] Downstream primer HIF-1α-PAS-B-:5'-GCC CTCGAG CTACAAGTCGTGCTGAATAATACCACT-3' (SEQ ID NO: 4)
[0026] BamH I and Xho I restriction sites were introduced into the underlined part, and HIF-1α-PAS- B gene (see SEQ ID NO: 5 (348bp) for the sequence of the target cloned gene, and see SEQ ID NO: 6 for the amino acid sequence expressed by the sequence of SEQ ID NO: 5), connected to pGEX-4T-1 (purchase pGEX-4T-1 In GE healthcare) expression vector, the gene sequence was determined to confirm the correctness of the obtained target gene sequence. (See figure 1 )
[0027...
Embodiment 2
[0029] Nanobody screening process against the PAS-B domain of HIF-1α:
[0030] (1) Coating antigen: prepare 100μg / mL GST_HIF-1α-PAS-B solution with coating buffer (0.05M carbonate buffer pH 9.6), and use coating buffer (0.05M carbonate buffer pH 9.6) to prepare 100 μg / mL GST solution; coat the immunotubes with the above two solutions respectively, and let stand overnight; the immunotube coated with GST_HIF-1α-PAS-B solution is called tube A, and the immunotube coated with GST solution called tube B;
[0031] (2) Blocking: the supernatant was discarded, and tubes A and B were washed three times with PBS (phosphate buffered saline) with a pH of 7.4. Add PBS solution containing 2% (g / ml) skimmed milk, let stand at 37°C for 3 hours, discard the supernatant, and wash tubes A and B three times each with PBST (phosphate buffered saline, containing 0.05% Tween-20) , and then wash tube A and tube B three times with PBS;
[0032](3) Pretreatment of Nanobody phage library: Take 300 μL...
Embodiment 3
[0040] A single positive clone specific for HIF-1α-PAS-B was screened by phage enzyme-linked immunosorbent assay (ELISA):
[0041] (1) Preparation of phage nanobodies: After the third round of step (7), the Escherichia coli TG1 (purchased from New England Biolabs) was plated, and 96 monoclonal bacteria containing phage were picked and inoculated into 96-well plates. , cultivate overnight at 37°C, add helper phage M13K07 at a ratio of M13K07:TG1=20:1, incubate at 37°C for 45min, centrifuge, discard the supernatant, resuspend the bacteria, and culture overnight. centrifuged, and the resulting supernatant was set aside;
[0042] (2) Coating: Coat 100 μg / mL GST_HIF-1α-PAS-B, 100 μL per well in a new 96-well plate, and let stand overnight at 4°C. Also coated with GST as a negative control;
[0043] (3) Blocking: Discard the coating solution and wash three times with PBS with a pH of 7.4. Add a PBS (pH 7.4) solution containing 2% skimmed milk to each well and let it stand for 2 h...
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