Plasmid combination and application thereof in preparing modified immune cells
Provided in the present disclosure is a method for using a four-plasmid system to prepare modified immune effector cells. The method comprises: forming a lentivirus by using four plasmids within 293T cells, extracting and obtaining the lentivirus, then transfecting immune effector cells by using the lentivirus, and expressing a chimeric antigen receptor. Also provided in the present disclosure is a use of the immune effector cell obtained by using the described method and of a composition containing the immune effector cell.
1. A plasmid combination, wherein the plasmid combination comprises plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev; and the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present at a ratio of 2-6: 1-1.5: 1-31-1.5;
wherein the plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor, and wherein the chimeric antigen receptor comprises an amino acid sequence shown in SEQ ID NO. 1.
2. The plasmid combination according to claim 1 , wherein the plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid molecule comprises a nucleic acid sequence shown in SEQ ID NO. 2.
3. A plasmid combination, wherein the plasmid combination comprises plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev; and the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present at a ratio of 11.8: 3.53: 6.33: 2.3, 13.8: 3.48: 5.31: 2.54 or 14: 4.67: 4.67: 4.67;
wherein the plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor, and wherein the chimeric antigen receptor comprises an amino acid sequence shown in SEQ ID NO. 1.
4. A method for preparing a lentiviral vector, the method comprising introducing the plasmid combination according to claim 1 into a cell.
5. The method according to claim 4 , wherein the cell is 293T.
6. A method for preparing a modified immune effector cell, comprising preparing the lentiviral vector according to the method according to claim 4 .
7. The method according to claim 6 , comprising introducing the lentiviral vector into an immune effector cell.
8. The method according to claim 7 , wherein the immune effector cell is selected from the group consisting of a T lymphocyte and a natural killer cell.
9. A modified immune effector cell prepared by the method according to claim 6 .
10. The modified immune effector cell according to claim 9 , wherein the chimeric antigen receptor is expressed on the surface of the immune effector cell.
11. A composition comprising the modified immune effector cell according to claim 9 .
12. The plasmid combination according to claim 1 , wherein the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present at a ratio of 3-6: 1-1.5: 1-3: 1-1.5.
13. The plasmid combination according to claim 1 , wherein the plasmid Seq1 does not comprise GFP gene.
14. The plasmid combination according to claim 1 , wherein the plasmid Seq1 does not comprise T2A gene.
15. The plasmid combination according to claim 1 , wherein the plasmid Seq1 does not comprise ampicillin resistance gene.
16. The plasmid combination according to claim 1 , wherein the plasmid Seq1 comprises kanamycin resistance gene.
17. The method according to claim 4 , wherein the cell is 293T/17.