CRISPR/Cas9技术构建sT2细胞系筛选猪源病毒SLA-I限制性CTL表位的研究

TAP基因在细胞内主要负责转运内源性抗原肽给MHC I，形成MHC I-peptides复合物递呈到细胞表面。如果将TAP基因缺失，将阻断内源性多肽的递呈，从而可以专门研究外源性多肽的递呈，以筛选病毒多肽表位。本研究以PK-15细胞为原料，通过CRSPR/Cas9技术敲除TAP1和TAP2基因，最终建立 TAP1和TAP2(-/-)PK-15(sT2)细胞。设计并合成猪源病毒 SLA-I 限制性多肽，分别负载sT2细胞和已转染外源SLA-I分子的 sT2 细胞，SLA-I 荧光标记单抗检测细胞表面SLA-I表达情况，与正常PK-15细胞比较，可间接检测细胞系及外源 SLA-I 所结合的病毒多肽表位。ELISPOT和Tetramer验证多肽表位的活性。本研究将建立SLA-I限制性猪源病毒CTL表位细胞系筛选平台，并突破细胞系本身SLA-I分子的限制性，为今后猪源病毒多表位疫苗的研制奠定基础。

TAP genes are mainly in charge of transporting endogenous peptides to MHC class I and then the MHC I-peptides complex will be formed followed by being presented to the surface of the cell membrane. If the TAP genes were deficient, the presentation for endogenous peptides will be blocked. Then, exogenous peptides can be used to study their presentation in cell, so the viral peptide epitope can be screened. In this research, the porcine kidney cell line PK-15 was selected as target materials to knock out TAP1 and TAP2 genes by using CRISPR/Cas 9, finally to construct a TAP1 and TAP2 (-/-) PK-15 (sT2) cell line. Peptide epitopes derived from swine virus restricted by SLA-I were predicted and synthesized, and then the peptides were pulsed onto normal sT2 cell line and special sT2 cell line with an exogenous SLA-I transferred. Detection of the SLA-I-peptides complexes expression on the cell membrane for sT2 cell lines and exogenous SLA-I transferred sT2 cell line by using SLA-I monoclonal antibody labeled with FITC, comparing to the normal PK-15 cell, the candidate CTL peptide epitopes derived from swine virus bound with SLA-I will be identified indirectly. Finally the peptides’ CTL activity will be detected with ELISPOT and Tetramer assay. This research will construct a cellular platform to screen SLA-I restricted CTL epitopes derived from swine virus and it will break out the restriction of SLA-I for cell itself. Furthermore, this research will pave the way to develop multi-epitopes vaccine to defend swine viral disease later.