ELISpot技术检测猪抗口蹄疫γ干扰素方法的建立  

Establishment of ELISpot Technique for Detecting IFN-γ against Foot and Mouth Disease Virus

作　　者：刘佳[1] 高习习 李巍[1] 程淑晶[1] 李广焱[1] 于汉勋[1] 王钦富[2] LIU Jia GAO Xi-xi LI Wei CHENG Shu-jing LI Guang-yan YU Han-xun WANG Qin-fu(Dalian Center for Animal Disease Prevention and Control, Dalian 116037, China College of Life Science and Technology , Dalian University, Dalian 116622, China)

机构地区：[1]大连市动物疫病预防控制中心,大连116037 [2]大连大学生命科学与技术学院,大连116622

出　　处：《中国畜牧兽医》2017年第1期53-58,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(81172777);大连市科技计划基金(2014B11NC096)

摘　　要：猪口蹄疫免疫防御是控制猪口蹄疫疫情的重要手段,免疫水平指标通常用免疫抗体水平判定。对于其细胞免疫水平检测尚缺乏成熟可靠的技术,本试验利用市售ELISpot试剂盒建立猪口蹄疫特异γ干扰素检测方法,刺激物分别采用疫苗全病毒颗粒(O/Mya98/XJ/2010)、口蹄疫病毒T细胞表位多肽池,以植物血凝素(PHA)为阳性对照,对细胞浓度、刺激物浓度、孵育时间等进行优化。优化条件为:外周血PBMC新鲜提取或冻存细胞成活率90%以上,最佳细胞数为2×105个/孔,最佳反应时间是16h,病毒粒子146S含量为100ng/mL,多肽最佳浓度10μg/mL,PHA的最佳浓度是20μg/mL。ELISpot技术检测口蹄疫病毒感染猪γ干扰素方法的建立,为口蹄疫免疫力评价及口蹄疫疫苗免疫效果评估及进一步研究其与疫苗保护力(PD50)的相关性奠定基础。The food and mouth disease virus（FMDV）vaccination is a key method in prevention and control of FMD,and the assay of antibodies to FMDV is always used to evaluate the immunity and preventive ability to FMD for its simplicity.However,the antibody titer is not always paralleled with the immunity to FMDV,for cellular immunity is also an important factor in FMDV response.So,we set up and optimized a ELISpot technique to detect specific interferon gamma（IFN-γ）to FMDV.Both the peptide pool and whole virus particle of FMDV（O/Mya98/XJ/2010）were used as stimulators,and the phytohaemagglutinin（PHA）was used as positive control.The final concentration of PBMC,whole virus and peptide pool and incubation time were optimized.The results showed that the optimized condition for ELISpot to detect specific IFN-γto FMDV were as followed,more than 90% viability of fresh isolated or cryopreserved PBMC were seeded at the suitable concentration of 2×105 cells/well,stimulated with whole virus particle containing 100ng/mL 146 Sor T cell epitope peptide pool consisting of 10μg/mL each peptide or 20μg/mL PHA and incubated for 16 hours.The foundation of ELISpot technique to detect specific IFN-γto FMDV would provide a valuable means to evaluate the cellular immunity to FMDV of swine inoculated with FMD vaccine.The relevance of specific IFN-γlevel to FMDV with50% preventive dose（PD50）of challenge test needed to be further investigated in the future.

关 键 词：ELISPOT 口蹄疫 Γ干扰素 

分 类 号：S852.65[农业科学—基础兽医学]