Polypeptide conjugate for targeting tumor cells as well as preparation method and application of polypeptide conjugate
A technology of polypeptide coupling and tumor cells, which is applied in the biological field and can solve difficult problems such as tumor antigens
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0049] Example 1, Construction of the polypeptide conjugate pHLIP-CD19
[0050] 1. Linking CD19 with coupling agent sulfo-SMCC
[0051] Dissolve 4800nmol sulfo-SMCC (Thermo Scientific, USA) in 300μL PBS solution and 10nmol CD19 (Sino Biological Inc, China) in 200μL PBS solution, mix the sulfo-SMCC solution and CD19 solution to 1.5mL The Eppendorf centrifuge tube was gently shaken at room temperature for 2 hours. During the whole process, the pH of the mixed solution was controlled to be 7.4 to obtain a CD19 polypeptide conjugate solution. Then, the CD19 polypeptide conjugate solution was purified with a column of NAP-5 (GE Healthcare, UK) pre-equilibrated with PBS buffer to obtain a solution containing the purified CD19 polypeptide conjugate, such as figure 1 Shown, a schematic diagram of the connection of the pHLIP-CD19 polypeptide conjugate.
[0052] 2. Linking CD19 polypeptide conjugates with the insertion peptide pHLIP targeting tumor cells
[0053] Such as image 3 Sh...
Embodiment 2
[0055] Example 2, Cell Transformation of pHLIP-CD19 Polypeptide Conjugates
[0056] 1. Transform the recombinant pHLIP-CD19 polypeptide conjugate to the cell surface.
[0057] OCI-LY8 is a human diffuse large B lymphoma cell line, which itself expresses CD19 molecules. It is used as a positive control group for killing experiments. It is cultured in RPMI-1640 cell culture medium. MG63 cells are osteosarcoma cells, cultured with DMEM medium , add 10% FBS (WISENT, Canada), culture at 37°C, 5% CO 2 in the cell culture incubator.
[0058] The pHLIP-CD19 polypeptide conjugate and MG63 cells were respectively co-cultured and induced for 2 hours under the environment of pH=6.8, during which the pH value of the solution was maintained. Such as figure 2 Shown is a schematic diagram of the process in which the pHLIP-CD19 polypeptide conjugate of this example undergoes self-curling and is inserted into the surface of the tumor cell membrane.
[0059] 2. Flow cytometry for peptide in...
Embodiment 3
[0061] Embodiment 3, the detection of cytotoxicity in vitro
[0062] 1. Construction of CAR T cells
[0063] T cells were isolated from human blood using the magnetic bead method. After being activated by the CD3 / CD28 complex, the packaged lentivirus was used to infect according to the virus titer, and the expression level was detected by flow cytometry for 48 hours.
[0064] Add 15ml of lymphocyte separation medium to a 50ml centrifuge tube, slowly add 30ml of whole blood into the 50ml centrifuge tube filled with lymphocyte separation medium, and centrifuge at 800g for 25min (centrifugation speed increases slowly, up to 1 down to 0). After centrifugation, carefully draw the mononuclear cell layer cells into a new 50ml centrifuge tube, add PBS to 50ml, gently pipette evenly, and centrifuge at 500g for 10min.
[0065] Resuspend the cell pellet with 50ml PBS and count the cells. Centrifuge at 300g for 10min. Cell pellets were resuspended in buffer (x-vivo basal medium + 10% F...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More