| Objective:Crispr-Cas9 gene editing technology is used to genetically modify tumor mutation sites to enhance the immunogenicity of tumor cells,so that immune cell therapy has a better effect on tumor cells with low antigenicity,and lays the foundation for tumor cell immunotherapy..Method:1.The full-length gene sequence encoding HLAAA0201 was cloned from the T2 cell c DNA,and then ligated to B2 M as a complete HLAA2 type MHC class I molecule encoding gene and ligated into a lentivirus expression vector.This vector was used to package the lentivirus.K562 cells expressing MHC class I molecules were introduced into HLAA2 type MHCI class molecules to make them have complete antigen presentation capabilities.BIMAS and SYFPEITHI were used to predict epitopes that are compatible with HLAA2 based on HBV surface proteins.At last,we tested the peptide-specific CTL against IFN-γsecretion of K562-A2 cells after loading peptides to verify whether K562 cells introduced with HLAA2 type MHCI molecules have the ability to present HLAA2-restricted antigens.2.Modification of the mutation site of TP53 gene in K562 cells by electroporation transfection to introduce antigenic epitopes derived from HBV.First,electroporation of K562 cells was performed by electroporation in terms of voltage,pulse time,and amount of plasmid.The optimized conditions were followed by transfection of the editing plasmids PX458 and Donor with optimized electrotransformation conditions,gene knock-in was verified by Junction PCR,and positive cells with red fluorescent markers were obtained by limiting dilution method.3.MHCI molecules and epitopes on the cell surface are bound by non-covalent bonds.In order to identify whether the epitope knocked in by Crispr-Cas9 is expressed and presented to the cell surface correctly,we wash the cells by means of weak acid elution.Surface peptide molecules,combined with UPLC / MS and difference comparison methods to verify the correct presentation of knock-in epitopes.In addition,by analyzing the signal-to-noise ratio of the intracellular enzymatic peptides of the knocked-in fragments,we can prove from the side that the epitope introduced by the genetic modification of the mutation site can be presented and different epitopes can be analyzed for specific Affinity of MHC molecules.4.Healthy people PBMC flow detection of HLA typing,HLAA2 monocytes were typed to induce mature DC cells in vitro through cytokine stimulation,and mature DC cells were loaded with epitope peptide HBs Ag335-343 peptide and HLAA2 PBMC co-stimulation The HBs Ag335-343 peptide-specific CTL was induced by culture.The calcein method was used to verify the killing of CTL on HBs Ag335-343peptide-loaded T2 cells,and then the peptide-specific CTL was used to target K562,K562-A2,and K562-A2 under different target ratios.-HBs Ag cells were tested for killing,the killing effect was verified by calcein method,and finally the HLAA2 antibody blocking test was used to verify whether the killing process was HLAA2 restricted.Result:1.The gene sequence encoding HLAA0201 type MHCI-like molecule was successfully cloned,and a lentivirus expression vector was constructed based on this vector.Based on this vector,the lentivirus was packaged and infected with K562 cells.Through drug screening and flow sorting,we obtained stable expression of HLAA2 K562 cells of type I MHC class I molecules,the growth curve of the cell line and the original K562 cell line were measured by the CCK8 method,and there was no significant difference in the growth status of the two cell lines.The IFN-γElisa test proved that K562-A2 has the restriction of presenting HLAA2 The function of epitope peptides lays the foundation for the correct presentation of subsequent gene knock-in epitopes.2.Optimizing the conditions for electroporation of K562 cell lines by electroporation with multiple electroporation transfection experiments.The macromolecular plasmid PX458 was able to transfect K562 cell lines with high efficiency(> 60%),and the HBs Ag335-343 coding sequence was successfully constructed.Co-transfection of PX458 and Donor into K562-A2 cell line with Mcherry-labeled Donor.By limiting dilution method we obtained the insertion of TP53 gene into HBs Ag335-343 coding sequence in K562-A2 cell line and K562-A2-HBs Ag cells in Mcherry.Line,Junction PCR sequencing results verified that the insertion site was correct.3.The results of full-wavelength scanning of the eluted samples showed significant differences in absorbance between 200 nm and 220 nm.It can be considered that some cell surface peptides were successfully extracted.After UPLC/Q-TOF MS detection,the K562-A2-HBs Ag sample group was specific at the position of the standard.Peaks appeared in the control group,K526 and K562-A2 groups,and no peaks appeared here,indicating that specific site knock-in antigens can be presented to the cell surface,and the signal-to-noise ratio results of the peptides also verified this view from the side.4.Flow phenotype experiments showed that mature DC cells were successfully isolated and induced.DCs successfully induced CTL from cell morphological changes.By inducing CTL killing of T2 cells loaded with HBs Ag335-343 peptide,it was known that induced CTLs.Peptide-specific,peptide-specific CTL killing target cell test results show that the K562-A2-HBs Ag target cell group has a higher cleavage rate on each target ratio than the K562 and K562-A2 target cell groups,and has passed The HLAA2 antibody blocking experiment proved that the killing of target cells by force peptide specific CTL is HLAA2 restricted Conclusion:The Crispr-Cas9 gene editing method is used to specifically target the mutation sites of tumor cells to introduce specific foreign epitope genes into the mutation sites,and the specific recognition of Crispr-Cas9 mutation sites is transformed into the immune system pair.The specific recognition of tumor cells,which significantly enhances the body’s immune system’s ability to recognize and kill tumor cells,is feasible.We believe this will be an important development direction of anti-tumor immunotherapy. |
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