猪全血中IFN-γ、IL-2、TNF-αTaqMan定量RT-PCR方法的建立及对猪瘟疫苗的免疫评价

Establishment of the TaqMan real-time RT-PCR for detecting IFN-γ,IL-2,and TNF-α from pig whole blood and assessment of cellular immune response in pigs vaccinated with classical swine fever vaccine

为建立猪相关细胞因子的检测方法,本研究针对GenBank中猪IFN-γ、IL-2、TNF-α的基因设计特异性引物和荧光探针,建立了这3种细胞因子TaqMan荧光定量RT-PCR检测方法。结果表明,建立的检测方法在101～109拷贝/μL模板范围内具有良好的线性关系,相关系数R2均高达0.999。利用该方法对猪瘟(CSF)活疫苗进行细胞免疫评价,结果显示,CSF疫苗免疫组猪瘟病毒(CSFV)刺激细胞相对于空白对照细胞,IFN-γ、IL-2、TNF-αmRNA表达量在免疫后3 d～10 d之间均显著升高(p<0.05),而IFN-γ在14 d仍保持高水平表达;对照组CSFV刺激细胞和空白对照细胞的3种细胞因子表达量均无明显差异(p>0.05)。以上结果表明,该方法具有高度的特异性、敏感性和重复性,对不同疫苗的细胞免疫反应评价是可靠的。

For detection of porcine cytokines from pig whole blood, the real time TaqMan RT-PCRs were developed using specific primers and fluorescent probe designed according to the genes of IFN-γ IL-2 and TNF-α in GenBank. The results showed that the established method had a linear dynamic range from 10^1 copies/ixL to 109 copies/μL, with the correlation coefficient R2 of 0.999. The assay was used to assess cellular immune response of classical swine fever （CSF） attenuated live vaccine in pigs, the results demonstrated that the IFN-γ IL-2 and TNF-α mRNA expressions were significantly increased from 3 to 10 days （p〈0.05）, respectively; However, the higher expression of IFN-γ lasted to 14 days when CSFV stimulated cells in comparing with unstimulated control cells in pigs post immunization with CSFV attenuated vaccine. Nevertheless, those mRNA expressions had no obvious difference between method was highly specific, lmmumzatlons CSFV stimulated cells and sensitive and reproducible control cells in unvaccinated pig group. which was reliable for assessment cellular The results suggested immune response for that the vaccine