真核表达hCD1d二聚体的纯化及其对iNKT细胞的刺激效应  

Purification of Eukaryotic CD1d Dimer and Its Stimulation Effect on iNKT Cells

作　　者：杨敬[1] 吕燕霞[1] 翁秀芳[1] 梁智辉[1] 吴雄文[1] Yang Jing;Lv Yanxia;Weng Xiufang(Department of Immunology,Basic Medical College,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院基础医学院免疫学系,武汉430030

出　　处：《华中科技大学学报（医学版）》2021年第1期21-26,66,共7页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.31570913)。

摘　　要：目的建立真核表达的人CD1d(hCD1d)二聚体的纯化方法,并制备人工抗原提呈细胞,研究其对iNKT细胞的刺激效应。方法提取pFastBacTMDual+[β2m+CD1d/IgG1-Fc]昆虫杆状病毒质粒并转染至sf9细胞中,收集病毒感染上清,通过ELISA法鉴定融合蛋白中人IgG-Fc、人CD1d和人β2m等组分并测定hCD1d二聚体浓度。探索保持hCD1d二聚体完整构象的亲和层析条件,对hCD1d二聚体进行纯化和浓缩。应用加载α-GalCer的hCD1d二聚体制备人工抗原提呈细胞刺激健康个体外周血单个核细胞(PBMC),检测Th1、Th2、Th17细胞因子分泌水平与iNKT细胞的扩增效率。结果收集的第4代病毒感染上清中目的蛋白表达量较高。抗Ig-Fc抗体ELISA、抗CD1d抗体与抗β2m抗体双抗夹心ELISA检测显示sf9细胞表达的hCD1d二聚体构象正确,两种方法检测的蛋白浓度分别为(1.05±0.20)μg/mL和(0.85±0.01)μg/mL。亲和层析纯化结果显示用pH2.5的0.1 mol/L柠檬酸可较好地实现hCD1d二聚体有效纯化。健康个体iNKT细胞经人工抗原提呈细胞刺激3 d后主要产生Th1型细胞因子,上清中IFN-γ、TNF-α含量分别为(1876.26±96.73)pg/mL和(1186.25±98.63)pg/mL,明显高于对照组(79.98±18.52)pg/mL和(29.96±4.43)pg/mL,差异具有统计学意义(均P<0.01),其余Th2型(IL-4)、Treg型(IL-10)和Th17型(IL-17)细胞因子未见明显升高,差异均无统计学意义(均P>0.05),与iNKT细胞接收刺激后分泌细胞因子谱基本一致。健康个体PBMC接受刺激后,iNKT细胞迅速扩增,刺激第7天iNKT细胞占PBMC(4.17±0.76)%,而未包被hCD1d二聚体的微球对照组iNKT细胞占PBMC的比例仅为(0.41±0.09)%。这些结果说明包被有hCD1d二聚体的人工抗原提呈细胞可特异性地刺激iNKT细胞分泌Th1型细胞因子并有效扩增。结论成功制备hCD1d二聚体,并建立基于人工抗原提呈细胞的iNKT细胞有效的刺激与扩增体系,为后续对iNKT细胞深入研究打下了基础。Objective To establish a method for purification of human CD1 d(hCD1 d)dimer expressed in eukaryotic cells and prepare artificial antigen-presenting cells to study their stimulation and expansion effects on iNKT cells.Methods pFastBacTMDual+[β2 m+CD1 d/IgG1-Fc]insect baculovirus plasmid was extracted and transfected into sf9 cells,and the virus infection supernatant was collected.The components of human IgG-Fc,human CD1 d and humanβ2 m in the fusion protein were identified by ELISA.Different eluting conditions were tried to set up optimized affinity chromatography method with high purity and complete conformation of hCD1 d dimer.Artificial antigen-presenting cells(aAPCs)were prepared by coatingα-GalCer-loaded hCD1 d dimer and anti-CD28 mAb on the microbeads to stimulate peripheral blood mononuclear cells(PBMC)in healthy individuals,followed by detection of Th1/Th2/Th17 cytokine levels and iNKT cell expansion rate.Results The supernatant of the fourth-generation virus infection had highest expression of the hCD1 d dimer and were selected for further test.Sandwich ELISA by anti-Ig-Fc antibodies or by antibodies against CD1 d andβ2 m was performed and showed(1.05±0.20)μg/mL and(0.85±0.01)μg/mL of hCD1 d dimer,respectively.Affinity chromatography purification showed that 0.1 mol/L citric acid with pH 2.5 maintained conformation of hCD1 d dimer and achieved effective purification.Human iNKT cells mainly produced Th1-type cytokines after stimulation byα-GalCer-loaded hCD1 d aAPCs for three days.The contents of IFN-γand TNF-αsupernatants were(1876.26±96.73)pg/mL and(1186.25±98.63)pg/mL,which were significantly higher than the control group(79.98±18.52)pg/mL and(29.96±4.43)pg/mL,with significant difference(P<0.01).The remaining Th2(IL-4),Treg(IL-10)and Th17(IL-17)cytokines were not significantly increased.The difference was not statistically significant(P>0.05).This is consistent with the spectrum of cytokines secreted form iNKT cells after receiving stimulation.In the experimental group,after healthy indi

关 键 词：hCD1d二聚体 亲和层析 Α-GALCER INKT细胞 细胞因子 

分 类 号：R392.12[医药卫生—免疫学]