IFN-γ-ELISpot方法用于评价猪伪狂犬病疫苗免疫效果

doi:10.11843/j.issn.0366-6964.2017.10.017

摘要/Abstract

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2 μg·孔^-1、外周血单核细胞（PBMC）浓度10⁶·孔^-1，培养时间36 h。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8 d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:

Due to the deficiency of antibody detection methods in evaluation of immunity against swine pseudorabies virus, to fully evaluate the cell-mediated immunity of PRV vaccine in combination with humoral immunity, we try to develop an Enzyme Linked Immuno-spot Assay (ELISpot) method for the detection of PRV specific IFN-γ level. In this study, according to the basic protocol of ELISpot, we tried to optimize the best conditions of stimulant concentration, peripheral blood mononuclear cells (PBMC) density and incubation time. After that, an animal experiment was performed, twenty PRV gB antibody-free piglets were divided into four groups which were administered with attenuated live vaccine, inactivated vaccine, live vaccine plus inactivated vaccine and normal saline (as control group). The immune response was evaluated by our in-house IFN-γ ELISpot,gB-ELISA and neutralization test, together with the results of challenge test. After optimization, the PRV IFN-γ ELISpot protocol was determined. The optimal stimulant concentration, cells density/well and incubation time were 2 μg·well^-1, 10⁶ cell·well^-1, and 36 h. Results obtained from the animal experiment revealed that live plus inactivated vaccine group showed the high level of both humoral and cellular immunity, the serum neutralization (SN) antibody titer was 1∶14.48 and the ELISpot result was 151.8±26.61, while those of SN antibody and ELISpot in live vaccine group and inactivated vaccine group were 1∶32.36, 40.4±9.07 and 1∶1.36, 189.6±27.36, respectively. The challenge test showed that the protective effect of live vaccine plus inactivatel vaccine was best as body temperature, viral shedding and clinical signs are the slighter in this group than those in other groups. The inhouse IFN-γ ELISpot results had a good relation with challenge test, providing an effective method for the evaluation of both cell immune response and optimal immune procedures for porcine pseudorabies vaccination.

中图分类号:

S858.285.3

谭鑫, 库旭钢, 于学祥, 郭恒科, 何东贤, 范盛先, 何启盖. IFN-γ-ELISpot方法用于评价猪伪狂犬病疫苗免疫效果[J]. 畜牧兽医学报, 2017, 48(10): 1939-1948.

TAN Xin, KU Xu-gang, YU Xue-xiang, GUO Heng-ke, HE Dong-xian, FAN Sheng-xian, HE Qi-gai. The IFN-γ-ELISpot for Evaluating the Immune Effect of Swine Pseudorabies Vaccine[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1939-1948.

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