General CAR-T cell and preparation method and application thereof

[0070] The construction of embodiment 1 plasmid

[0071] 1. The plasmids constructed in this example include

[0072] LV-EF-1a-CD19CAR(4-1BB)-mCherry (positive control, see image 3 )

[0073] AAV6-TCR-CD19CAR (4-1BB) (experimental vector, see the structure diagram figure 1 , 4 )

[0074] AAV6-TCR-GFP (negative control, see figure 2 )

[0075] 2. Plasmid construction and virus packaging process

[0076] 2.1 LV-EF-1a-CD19CAT(4-1BB)-mCherry plasmid construction

[0077]The sequence is shown in SEQ ID NO:10.

[0078] Obtain LV-EF-1a-CD19CAR(4-1BB)-mCherry plasmid

[0079] First construct the plasmid map on ApE or Snapgene software.

[0080] The functional region of the plasmid: LTR-EF-1a-CD19CAT(4-1BB)-mCherry-LTR for gene synthesis.

[0081] The sequence was cloned into a lentiviral vector plasmid.

[0082] Plasmid transformation, smearing, picking clones, extracting plasmids (minor extraction), confirming correctness by sequencing, extracting plasmids again (major...

[0097] Embodiment 2 virus packaging process

[0098] 2.1 LV packaging

[0099] Prepare DMEM+10%FBS medium+antibiotics

[0100] Resuscitated 293T cells, subcultured, and judged that the cells were in good condition

[0101] The day before encapsulation, divide into 6 10cm culture dishes, each seed 2.5M cells, after 24 hours reach 70% confluence and then encapsulate.

[0102] Infection plasmids include: pSLQ5367, pCMV-dR8.91, pMD2-G, TransIT-Virus Reagent, Opti MEM.

[0103] The specific ratio is in accordance with the 10-cm dish in Table 1.

[0104] Table 1 Condition ratio of DNA transfection

[0105]

[0106] 2.2. AAV6 virus packaging

[0107] Taking T75flask as an example, the number of cells was 9M-10M when passaged in the afternoon of the previous day

[0108] Reagent name Reagent quantity

[0109] Vector plasmid 7.5ul (1.0ug / ul)

[0110] Packaging plasmid 7.5ul (1.0ug / ul)

[0111] Helper plasmid 7.5ul (1.0ug / ul)

[0112] OptiMEM 1.5ml

[0113] TanslT-VirusG...

[0116] Example 3 T cell sorting

[0117] 1. T cell sorting

[0118] 1. Obtain a blood sample (at least 50 mL) from a healthy donor. Eligible patients who have been tested for the following diseases (not limited to these tests). Including: hepatitis A, hepatitis B, hepatitis C, AIDS, syphilis antibodies, tuberculosis, genetic diseases, etc.

[0119] 2. Obtain peripheral blood mononuclear cells (PBMC)

[0120] 1) Mix Ficoll-PaqueTM PLUS (#07957) evenly and place in an empty tube;

[0121] 2) Dilute the blood sample (2X) with PBS+2% FBS;

[0122] 3) Place the blood sample in step 2) on the upper layer of the solution in step 1) to minimize the mixing of blood and Ficoll-PaqueTM PLUS, and centrifuge at 400g for 30 minutes at room temperature and then slow down naturally;

[0123] 4) Discard the upper layer of plasma after centrifugation in step 3), and take the interface layer between the plasma and Ficoll-PaqueTM PLUS solution, which is the peripheral blood mononuclear cells...