Anti-CTLA-4 nanobody Nb36 as well as preparation method and application thereof
A technology of CTLA-4 and nano-antibody, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, nanotechnology for materials and surface science, etc., which can solve difficult large-scale production, high production cost, and limited application range and other issues
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0093] Embodiment 1, the preparation of nanobody
[0094] The present invention provides four nanobodies derived from camels, whose names are respectively Nb16, Nb30, Nb36 and Nb91. Wherein, the amino acid sequence of Nb16 is shown in SEQ ID No.1 in the sequence listing, encoded by the nucleotide sequence of SEQ ID No.2; the amino acid sequence of Nb30 is shown in SEQ ID No.3 in the sequence listing, encoded by SEQ ID No. The nucleotide sequence encoding of No.4; the amino acid sequence of Nb36 is as shown in SEQ ID No.5 in the sequence listing, encoded by the nucleotide sequence of SEQ ID No.6; the amino acid sequence of Nb91 is as shown in SEQ ID No in the sequence listing .7, encoded by the nucleotide sequence of SEQ ID No.8.
[0095] 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.2, and keep the other sequences unchanged to obtain the recombinant vector pComb3-Nb16. ...
Embodiment 2
[0108] Example 2, Determination of Nanobody and CTLA-4 Binding Rate
[0109] 1. Determination of the binding rate of Nanobody to CTLA-4 (direct method)
[0110] T cells were isolated from peripheral blood cells of healthy volunteers and cultured in 96-well plates. After adding 10 μg / mL PHA (sigma, L9017) to T cells for co-culture for 72 h, Nb16 (1 μg) from Example 1 was added to 1×10 6 Incubate the above T cells in the dark at 4°C for 30 min, wash with PBS three times, add 1 μg PE anti-HA tag antibody (abcam, Clone: 16B12) and incubate at 4°C for 30 min, wash with PBS three times, put the sample on the BACKMAN flow cytometer , the result is as Figure 5 Shown in A.
[0111] According to the above method, Nb16 was replaced by Nb30, Nb36 and Nb91 respectively, and other steps were kept unchanged, and the binding results of Nb30, Nb36 and Nb91 and CTLA-4 were obtained, respectively as follows Image 6 Middle A, Figure 7 Middle A, Figure 8 Shown in A.
[0112] 2. Determi...
PUM
| Property | Measurement | Unit |
|---|---|---|
| diameter | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More