CAJ | 학술논문

目的探讨酶联免疫斑点试验(ELISPOT)检测人博卡病毒(HBoV) VP2病毒样颗粒(VLPs)诱导特异性细胞免疫反应的最佳条件.方法 HBoV VP2 VLPs免疫小鼠后,用ELISPOT方法检测小鼠的特异性细胞免疫反应,观察不同多肽刺激、不同细胞培养时间、不同细胞浓度以及不同浓度特异性刺激多肽条件下ELISPOT结果.结果多肽P3(GYIPIENEL)及P5(LYQMPFFLL)刺激HBoV1 VLPs免疫小鼠脾脏淋巴细胞产生斑点数分别为233个/10(6)和157个/10(6)细胞,P8(GYIPVIHEL)刺激HBoV2 VLPs免疫小鼠脾脏淋巴细胞产生斑点数为113个/10(6)细胞；培养时间以24 h为最佳,此时HBoV1与HBoV2实验组特异性分泌IFN-(ｒ)的比率分别为232个/10(6)和119个/10(6)细胞；小鼠脾脏淋巴细胞浓度以5×10(5)为最佳,此时HBoV1与HBoV2实验组特异性分泌IFN-(γ)的比率分别为232个/10(6)和108个/10(6)细胞；刺激物浓度10μg/ml为最佳,HBoV1与HBoV2实验组特异性分泌IFN-(γ)的比率分别为233个/10(6)和96个/10(6)细胞.结论 HBoV1与HBoV2特异性BABL/c小鼠T细胞表位多肽分别为HBoV1:P3；HBoV2:P8.检测人博卡病毒VP2病毒样颗粒诱导特异性细胞免疫反应ELISPOT方法最佳实验条件为:培养时间24 h,细胞浓度5×10(5)细胞/孔,刺激多肽终浓度10 μg/ml,可用于HBoV在小鼠上的细胞免疫研究.
목적 탐토매련면역반점시험(ELISPOT)검측인박잡병독(HBoV) VP2병독양과립(VLPs)유도특이성세포면역반응적최가조건.방법 HBoV VP2 VLPs면역소서후,용ELISPOT방법검측소서적특이성세포면역반응,관찰불동다태자격、불동세포배양시간、불동세포농도이급불동농도특이성자격다태조건하ELISPOT결과.결과 다태P3(GYIPIENEL)급P5(LYQMPFFLL)자격HBoV1 VLPs면역소서비장림파세포산생반점수분별위233개/10(6)화157개/10(6)세포,P8(GYIPVIHEL)자격HBoV2 VLPs면역소서비장림파세포산생반점수위113개/10(6)세포；배양시간이24 h위최가,차시HBoV1여HBoV2실험조특이성분비IFN-(ｒ)적비솔분별위232개/10(6)화119개/10(6)세포；소서비장림파세포농도이5×10(5)위최가,차시HBoV1여HBoV2실험조특이성분비IFN-(γ)적비솔분별위232개/10(6)화108개/10(6)세포；자격물농도10μg/ml위최가,HBoV1여HBoV2실험조특이성분비IFN-(γ)적비솔분별위233개/10(6)화96개/10(6)세포.결론 HBoV1여HBoV2특이성BABL/c소서T세포표위다태분별위HBoV1:P3；HBoV2:P8.검측인박잡병독VP2병독양과립유도특이성세포면역반응ELISPOT방법최가실험조건위:배양시간24 h,세포농도5×10(5)세포/공,자격다태종농도10 μg/ml,가용우HBoV재소서상적세포면역연구.
Objective To discuss the enzyme linked immune spot test (ELISPOT) detected the cellular immune response induced by human Bocavirus(HBoV) VP2 virus-like particles(VLPs).Methods After immunized by HBoV VP2 VLPs,the specific cellular immune response in mice were detected by ELISPOT assay,observe the ELISPOT results at the conditions of different polypeptide stimulate,different cell culture time,different cell concentration and different specific stimulus peptide concentration,then screening the right ELISPOT experimental conditions and establish the ELISPOT method.Results The spots induced by HBoV1 VLPs immunized mice spleen lymphocytes stimulate with polypeptide P3 (GYIPIENEL) and P5 (LYQMPFFLL)were 233 spots/10(6) cells and 157 spots/10(6) cells,spots induced by HBoV2 VLPs immunized mice spleen lymphocytes stimulate with polypeptide P8 (GYIPVIHEL)were 113 spots/10(6) cells; 24 hours is the best time for culture,at this time HBoV1 and HBoV2 groups specificity secretion IFN-gamma ratio were 232 spots/10(6) cells and 119/10(6) cells; Best concentration of mice spleen lymphocyte is 5 × 10(5),right now HBoV1 and HBoV2 group specificity secretion IFN-gamma ratio were 232 spots/10(6) cells and 108/10(6) cells; Best concentration of polypeptides is 10 μg/ml,HBoV1 and HBoV2 group specificity secretion IFN-gamma ratio were 233 spots/10(6) cells and 96/10(6) cells.Conclusions HBoV1 and HBoV2 specificT-cell epitope in BABL/c mice were P3,P5 (HBoV1)and P8 (HBoV2).The best experiment condition were:cell cultivated for 24 h,cells concentration for 5 × 10(5) cells/well,stimulating polyperides concentration for 10 μg/ml,it can use to study the cellular immune induced by HBoV in mice.