鸭IFN-γ,IL-2 mRNA荧光定量RT-PCR方法的建立及应用

Establishment and application of a real-time assay for detecting of IFN-γ and IL-2 mRNA in ducks

为评价疫苗免疫对鸭细胞免疫应答反应的影响,本研究提取鸭脾脏组织总RN A,采用鸭IFN-γ、IL-2、GAPDH基因特异性引物,建立了SYBR Green I荧光定量RT-PCR(qRT-PCR)方法。结果显示IFN-γ、IL-2、GAPD H基因扩增标准曲线相关系数均高于0.99;3者扩增效率相近,均接近于100%;融解曲线呈特异性单峰;各基因组内和组间变异系数均小于2%。应用该方法检测不同佐剂禽流感灭活疫苗免疫樱桃谷鸭后脾脏组织IFN-γ和IL-2 mRNA的相对表达水平。结合免疫后抗体水平及攻毒保护结果,比较不同佐剂对禽流感灭活疫苗免疫应答的影响。结果显示,金黄色葡萄球菌毒素A重组蛋白(rSEA)佐剂组IFN-γ和IL-2 mRNA表达水平显著高于其他免疫组(p<0.05)。结果表明,建立的qRT-PCR特异性、重复性和稳定性良好,能够快速、准确检测鸭体内细胞因子的表达水平,为进一步评价疫苗免疫效果奠定了基础。

To evaluate the cellular immune responses induced by vaccination in duck, one-step SYBR Green Ⅰ real-time RT-PCR method was established using specific primer targeting conserved sequences of IFN-γ IL-2 and GAPDH genes. The results showed correlation coefficient of relative standard curves for the amplification of the three genes were more than 0.99, with a single peak in the melting curves. Moreover, amplification efficiency of the three genes was similar and closed to 100%, with intra-and inter-assay coefficients of variations were less than 2%. The mRNA expression levels of IFN-γ and IL-2 were detected with this method in the spleen of Cheery Valley ducks immunized with inactivated AIV vaccine with different adjuvant. The results showed mRNA expression levels of the IFN-γ and IL-2 were significant higher in the recombinant staphylcococcal enterotoxin A （rSEA） adjuvant group than that in other groups （p〈0.05）. Our study indicated that the real-time PCR could be used for rapid detection of cytokines for evaluation of the celluar immune responses in duck.