ELISpot技术检测猪抗口蹄疫γ干扰素方法的建立

doi:10.16431/j.cnki.1671-7236.2017.01.007

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (1): 53-58.doi: 10.16431/j.cnki.1671-7236.2017.01.007

ELISpot技术检测猪抗口蹄疫γ干扰素方法的建立

刘佳¹, 高习习², 李巍¹, 程淑晶¹, 李广焱¹, 于汉勋¹, 王钦富²

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

收稿日期:2016-05-16 出版日期:2017-01-20 发布日期:2017-01-19
通讯作者: 王钦富 E-mail:qinfuwang@126.com
作者简介:刘佳(1985-),女,黑龙江勃利人,硕士,兽医师,研究方向:动物疫病检测,E-mail:liujia_0228@163.com
基金资助:
国家自然科学基金（81172777）；大连市科技计划基金（2014B11NC096）

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

LIU Jia¹, GAO Xi-xi², LI Wei¹, CHENG Shu-jing¹, LI Guang-yan¹, YU Han-xun¹, WANG Qin-fu²

1. Dalian Center for Animal Disease Prevention and Control, Dalian 116037, China;
2. College of Life Science and Technology, Dalian University, Dalian 116622, China

Received:2016-05-16 Online:2017-01-20 Published:2017-01-19

摘要/Abstract

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16 h，病毒粒子146 S含量为100 ng/mL，多肽最佳浓度10 μg/mL，PHA的最佳浓度是20 μg/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。

关键词: ELISpot; 口蹄疫; γ 干扰素

Abstract:

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-γ to 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 100 ng/mL 146 S or T cell epitope peptide pool consisting of 10 μg/mL each peptide or 20 μg/mL PHA and incubated for 16 hours. The foundation of ELISpot technique to detect specific IFN-γ to FMDV would provide a valuable means to evaluate the cellular immunity to FMDV of swine inoculated with FMD vaccine. The relevance of specific IFN-γ level to FMDV with 50% preventive dose (PD₅₀) of challenge test needed to be further investigated in the future.

Key words: ELISpot; foot and mouth disease; IFN-γ

中图分类号:

S852.65

刘佳, 高习习, 李巍, 程淑晶, 李广焱, 于汉勋, 王钦富. ELISpot技术检测猪抗口蹄疫γ干扰素方法的建立[J]. 《中国畜牧兽医》, 2017, 44(1): 53-58.

LIU Jia, GAO Xi-xi, LI Wei, CHENG Shu-jing, LI Guang-yan, YU Han-xun, WANG Qin-fu. Establishment of ELISpot Technique for Detecting IFN-γ against Foot and Mouth Disease Virus[J]. , 2017, 44(1): 53-58.

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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

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