Protein SaCas9 antigen epitope and application thereof in gene editing
An epitope, protein technology, applied in application, gene therapy, genetic engineering, etc., can solve problems such as liver injury and CD8+ T cell aggregation
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Embodiment 1
[0056] Example 1. Prediction of SaCas9 protein B cell antigen epitope
[0057] First, the amino acid sequence and crystal structure model of the SaCas9 protein (PDB: 5AXW) were retrieved from the RCSB PDB protein database, and then the sequence and structure model were input into the online prediction platform of IEDB ELLIPRO and DISCOTOPE 2.0 protein B cell epitopes.
[0058] ELLIPRO scores each amino acid residue based on continuous (linear) epitopes and discontinuous (conformational) epitopes. Carry out PI (Protrusion Index, prominence index) score to each predicted epitope, adopt the default setting with PI=0.5 as the threshold value, that is, residues with a protruding index higher than 50% will be identified as epitopes; use the default setting by residue distance As a threshold, that is, the distance is less than residues can be identified as a discontinuous epitope, thus 441 possible linear epitopes and 499 possible conformational epitopes were obtained respectivel...
Embodiment 2
[0062] Example 2. Rapid detection of mutant SaCas9 protein cleavage activity
[0063] Combining the parameter values of each prediction software, and excluding the amino acid residues of the cleavage functional domain and the reported functional sites, 23 candidate B cell epitope amino acid residues with the best prediction results were obtained. Amino acids at 23 sites were mutated by point mutation method. In order to reduce the impact on other properties of the protein on the basis of changing its antigenicity, all of them were mutated into neutral glycine or alanine with the smallest group acid.
[0064] Use the pre-constructed SaCas9 protein cleavage activity rapid detection system, which is a Hela cell line stably expressing luciferase, express the mutant SaCas9 to be detected in series with the sgRNA targeting luciferase, and use the empty plasmid transfection group as a control , the cleavage activity of 23 mutant SaCas9 proteins with single amino acid mutations in ...
Embodiment 3
[0065] Example 3. Verification of SaCas9 protein B cell antigen epitope
[0066] According to the predicted candidate results of SaCas9 protein B cell antigen epitopes, combined with the rapid detection results of mutant SaCas9 protein cleavage activity, the expression plasmids of 17 B cell antigen epitope single amino acid mutant SaCas9 proteins with unaffected cleavage activity were transiently transfected HEK293-17 cell line. 2d-3d after transfection, the cells were lysed to extract proteins, and the whole protein after cell lysis was purified by IP using Anti-Flag-Magnetic Beads, and finally 17 candidate B cell antigen epitope single amino acid mutant SaCas9 proteins were obtained.
[0067] After the 17 SaCas9 protein mutants and wild-type SaCas9 protein were quantified, they were coated on ELISA plates in equal amounts as antigens. Such as image 3 As shown, using the serum positive for SaCas9 protein-specific IgG antibody level screened from the Chinese population cohort...
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