Anti-IL13Ra2 nano antibody and application thereof
A nanobody, chimeric antigen receptor technology, applied in the field of biomedicine
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Embodiment 1
[0110] In this example, a phage nanobody library was constructed and panned, and ELISA was used for preliminary screening. The specific steps are as follows:
[0111] (1) Construction of phage nanobody library
[0112] The recombinant protein of IL13Ra2 extracellular segment was used to immunize alpaca, and after the serum titer was detected by ELISA, peripheral blood was drawn; lymphocytes were separated, total RNA was extracted, and then reverse-transcribed into cDNA, and then the VHH gene was amplified by nested PCR; the VHH gene Insert pShort phagemid, electroporation Competent cells, after amplification, separated and purified phages by PEG8000 / NaCI precipitation method to obtain antibody library; adjusted the concentration, subpackaged and frozen in -80°C refrigerator for later use;
[0113] (2) Screening of phage nanobody library
[0114] First, take 293T cells and incubate with the antibody library for negative screening, then take the supernatant and incubate with ...
Embodiment 2
[0116] In this example, the anti-IL13Ra2 nanobody (VHH-mIgG2a Fc nanobody) screened in Example 1 was expressed and purified, and the antibody affinity was measured. In order to further identify the antibodies obtained through screening, it is necessary to express the antibodies through mammalian cells. Therefore, a plasmid vector expressing VHH with a mouse Fc tag was constructed first, which was denoted as C-4pCP.Stuffer-mCg2a-FC. The specific steps are as follows :
[0117] (1) Use PCR to amplify the VHH fragment, the reaction system is as shown in Table 1, and the amplification program is as shown in Table 2 below;
[0118] Table 1
[0119] Table 2
[0120]
[0121]
[0122] (2) The enzyme digestion system is shown in Table 3, the enzyme digestion temperature is 37°C, the time is 6h, and the vector after enzyme digestion is used PCR purification kit for purification, the recovered DNA was dissolved in 45 μL water, and the concentration of DNA was detected;
[012...
Embodiment 3
[0143] In this example, flow cytometry was performed on the anti-IL13Ra2 nanobody of the example.
[0144] 293T (IL13Ra2 negative, IL13Ra2 - ), 293T-Mesothelin cells were mixed with the purified anti-IL13Ra2 nanobody, incubated in ice bath for 30min, then incubated with APC-labeled goat anti-mouse IgG antibody for 30min, and detected by flow cytometry, the results were as follows: figure 2 As shown, it shows that the anti-IL13Ra2 nanobody of the present invention can recognize the IL13Ra2 antigen on the cell surface.
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