CD44 antibody, chimeric antigen receptor and application thereof

[0076] This example is used to illustrate the preparation of CD44 antibody.

[0077] 1. Culture hybridoma cells

[0078] After recovering the hybridoma cell lines, culture them until the number of cells expands to about 1×10 7 At 1000rpm×5min, the cells were collected by centrifugation.

[0079] 2. Extraction of cellular RNA

[0080] In an ultra-clean workbench environment, add 1ml Trizol reagent to the centrifuged cells, let it stand for 5min, add 2mL of chloroform, shake vigorously for 15sec, let stand at room temperature for 3min, 12000rpm×15min, transfer the upper water sample layer to a new EP tube, add 0.5 mL isopropanol, let stand at room temperature for 10min. 12000rpm×10min. Discard the supernatant, add 1mL 75% ethanol, 7500rpmx5min, dry the precipitate, add 50μL double distilled water. The purity was identified and quantified by agarose electrophoresis, and stored at -70°C for future use.

[0081] 3. Prepare cDNA by reverse transcription

[0082] 1 μL total cellu

[0092] This example is used to illustrate the construction of a chimeric antigen receptor expression vector.

[0093] (1) Using the full sequence synthesis method, synthesize the nucleotide sequences shown in Table 1 respectively:

[0094] Table 1

[0095]

[0096] (2) Using pLVX-IRES-ΔNGFR (purchased from Clontech, Cat. No. 631982) as a vector, inserting the two nucleotide sequences (SEQ ID NO: 11 and 15) synthesized in step (1) respectively by conventional methods, Two kinds of lentiviral expression vectors of this example were obtained. At the same time, conventional methods were used to construct a plasmid overexpression vector containing SEQ ID NO:13.

[0098] This example is used to illustrate the binding activity of the recombinant anti-CD44 single chain antibody in this case to glioma tumor stem cells.

[0099] (1) The plasmid overexpression vector constructed in Example 2 was used for transfection, amplification and protein purification of Escherichia coli according to the following methods.

[0100] (2)Protein expression and purification process:

[0101] Transform the constructed plasmid into BL21 DE3 competent cells, inoculate the resistant LB plate medium, and grow overnight; select 6 single clones on the transformed plate, and inoculate 3ml of resistant liquid medium respectively; culture at 37°C, 220RPM until OD600nm 0.5- 0.6, add 0.5mM IPTG to induce expression at 20°C for 3.5 hours; collect the bacteria by centrifugation, sonicate, and detect the expression by SDS-PAGE. Analysis of small sample expression results: the protein is expressed in both the supernatant and the inclusion body, and soluble expression and pur