IFN-γ-ELISpot方法用于评价猪伪狂犬病疫苗免疫效果

发布时间：2018-06-28 17:05

本文选题：猪伪狂犬病 + IFN-γ-ELISpot　；参考：《畜牧兽医学报》2017年10期

【摘要】：鉴于常用的抗体检测方法不能完整反映免疫猪获得针对猪伪狂犬病毒的保护力,有必要建立猪伪狂犬病病毒(PRV)特异性IFN-γ的酶联免疫斑点检测(ELISpot)方法,并评估伪狂犬病疫苗细胞免疫的免疫效果。依照ELISpot基本操作流程,摸索PRV作为刺激原的最适浓度、外周血单个核细胞(PBMC)密度和作用时间以建立并优化该方法。将20头gB抗体检测阴性猪,随机分为单独弱毒疫苗免疫组、单独灭活疫苗免疫组、弱毒疫苗-灭活疫苗联合组及对照组,免疫后攻毒。利用所建方法,结合gB-ELISA、中和试验及攻毒保护试验,评价上述三种免疫程序的效果。试验结果表明:IFN-γ-ELISpot最佳条件为刺激原浓度2μg·孔~(-1)、外周血单核细胞(PBMC)浓度106·孔~(-1),培养时间36h。一免弱毒疫苗二免灭活疫苗组二免后2周,试验猪中和抗体效价可达1∶14.48,ELISpot斑点数可达(151.8±26.61)个·孔~(-1),且该免疫程序下,试验猪攻毒后体温、鼻拭子排毒及临床症状均低于其余试验组,攻毒8d后仅表现呼吸道症状;单独灭活疫苗免疫组二免后2周,试验猪的中和抗体效价可达1∶32.36,而ELISpot斑点数仅为(40.4±9.07)个·孔~(-1);单独弱毒疫苗免疫组二免后2周,试验猪的中和抗体效价仅为1∶1.36,但ELISpot斑点数可达(189.6±27.36)个·孔~(-1)。综合分析攻毒试验结果和ELISpot斑点数之间的相关性,本试验所建立的IFN-γ-ELISpot可用于猪伪狂犬病疫苗的细胞免疫评估,为免疫程序的评价和选择提供有效手段。
[Abstract]:Since the commonly used antibody detection methods can not completely reflect the protective power of immunized pigs against porcine pseudorabies virus (PRV), it is necessary to establish an enzyme-linked immunospot assay (Elispot) for porcine pseudorabies virus (PRV) specific IFN- 纬. The immune effect of pseudorabies vaccine was evaluated. According to the basic operating procedure of Elispot, the optimal concentration of PRV as stimulator, the density and duration of peripheral blood mononuclear cells (PBMC) were explored to establish and optimize the method. Twenty GB antibody negative pigs were randomly divided into three groups: single attenuated vaccine immunization group, single inactivated vaccine immunization group, combined attenuated vaccine and inactivated vaccine group and control group. The effects of the above three immune programs were evaluated by using the established method combined with the gB-ELISA-neutralization test and the anti-drug protection test. The results showed that the optimal conditions were the stimulation concentration of 2 渭 g pore ~ (-1), the concentration of peripheral blood monocyte (PBMC) of 106 holes ~ (-1) and the culture time of 36 h. The neutralizing antibody titer of the test pigs reached 1: 14.48% Elispot spots (151.8 卤26.61) holes ~ (-1) 2 weeks after the second immunization of the first attenuated vaccine and the inactivated vaccine group, and the body temperature of the tested pigs after the attack of the virus was (151.8 卤26.61) holes. The detoxification and clinical symptoms of nasal swabs were lower than those of the other test groups, and only respiratory symptoms were observed 8 days after exposure, and 2 weeks after immunization with inactivated vaccine alone. The neutralizing antibody titer of test pigs was 1: 32.36, but the number of Elispot spots was (40.4 卤9.07) holes ~ (-1), and the neutralizing antibody titer of test pigs was only 1: 1.36 two weeks after immunization with attenuated vaccine, but the number of Elispot spots was (189.6 卤27.36) holes ~ (-1). Based on the comprehensive analysis of the correlation between the results of anti-virus test and the number of Elispot spots, the IFN- 纬 -ELISpot established in this study can be used to evaluate the cellular immunity of porcine pseudorabies vaccine and provide an effective means for the evaluation and selection of immunization program.
【作者单位】：华中农业大学农业微生物学国家重点实验室;华中农业大学动物医学院;生猪健康养殖协同创新中心;
【基金】：国家生猪产业技术体系(CARS-35)
【分类号】：S858.28

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