Anti-PD-1 nanobody PD-1/nb18 and its preparation method and application
A nano-antibody and PD-1 technology, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of high production cost, poor stability and easy degradation, long development cycle, etc.
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Embodiment 1
[0074] Embodiment 1, the preparation of nanobody
[0075] The present invention provides a nanobody derived from camels, its name is PD-1 / Nb18, the amino acid sequence of the nanobody PD-1 / Nb18 is shown in SEQ ID No.8 in the sequence listing, represented by SEQ ID No. 9 nucleotide sequence encoding.
[0076] The nucleotide electrophoresis pattern of nanobody PD-1 / Nb18 is as follows figure 1 As shown, the first channel is a 2000bp molecular marker, the second channel is a PCR product, and the PCR product band is about 400bp.
[0077] Replace the DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (Biovector product) with the DNA molecule shown in SEQID No.9, and keep the other sequences unchanged to obtain the recombinant vector pComb3-PD-1 / Nb18, pComb3-PD- The only difference between 1 / Nb18 and pComb3 is that the DNA segment between the PstI and NotI recognition sequences of pComb3 is replaced by the DNA molecule shown in SEQ ID No.9. The reco...
Embodiment 2
[0085] Example 2, Determination of the Binding Rate of Nanobody PD-1 / Nb18 and PD-1
[0086] Determination of the binding rate of nanobody PD-1 / Nb18 to PD-1 (direct method)
[0087] 293T cells stably transfected with PD-1 were used to detect the binding rate of nanobody PD-1 / Nb18 to PD-1, and the nanobody PD-1 / Nb18 (1 μg) in Example 1 was added to 1-6×10 6 The above-mentioned 293T cells were incubated at 4°C in the dark for 20-40min, washed twice with PBS, added 5 μl PE anti-HA tag antibody (abcam, Clone: 20B12) and incubated at 4°C for 20-40min, washed twice with PBS, and the samples were On the BACKMAN flow cytometer, the results are as follows Figure 3B As shown, 293T cells not transfected with PD-1 were used as a control such as Figure 3A shown. Figure 3A is the binding percentage of blank control and PD-1 Nanobody PD-1 / Nb18 to 293T cells not transfected with PD-1; Figure 3B is the binding percentage of blank control and PD-1 nanobody PD-1 / Nb18 respectively stably...
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