人博卡病毒VP2病毒样颗粒诱导特异性细胞免疫反应的研究  

Enzyme-linked immunospot test detected specific cellular immune response induced by human Bocavirus VP2 virus-like particles

作　　者：邓中华[1] 谢志萍[2] 姚立红[2] 谢乐云[1] 李金松[2] 张兵[1] 段招军[2] 曹友德[1] 

机构地区：[1]湖南师范大学第一附属医院,长沙410006 [2]中国疾病预防控制中心病毒病预防控制所

出　　处：《中华实验和临床病毒学杂志》2013年第2期141-143,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：湖南省科技厅科研条件创新专项计划（项目编号：2012TT2004）

摘　　要：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HBoVVP2VLPs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HBoVlVLPs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。细胞，P8（GYIPVIHEL）刺激HBoV2VLPs免疫小鼠脾脏淋巴细胞产生斑点数为113个／10。细胞；培养时间以24h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HBoVl与HBoV2实验组特异性分泌IFN-γ的比率分别为232个／10。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。和96个／10^6细胞。结论HBoVl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24h，细胞浓度5×10^5细胞／孔，刺激多肽终浓度10~g／ml，可用于HBoV在小鼠上的细胞免疫研究。Objective To discuss the enzyme linked immune spot test （EL1SPOT） 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/106 cells and 157 spots/106 cells,spots induced by HBoV2 VLPs immunized mice spleen lymphocytes stimulate with polypeptide P8 （GYIPVIHEL）were 113 spots/10s 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 119/106 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 108/106 cells; Best concentration of potypeptides is 10 10^6g/ml,HBoV1 and HBoV2 group specificity secretion IFN -gamma ratio were 233 spots/10^6 cells and 96/106 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, ceils concentration for 5 ×10^5 cells/well, stimulating polyperides concentration for 10 10^6g/ml,it can use to study the cellular immune induced by HBoV in mice.

关 键 词：酶联免疫吸附测定 博卡病毒 人 免疫 细胞 

分 类 号：R3[医药卫生—基础医学]