外周血单个核细胞IFN-γ mRNA的测定及应用

Quantitation of IFN-γ mRNA in duck PBMC and its application

目的 研究鸭IFN γ(DuIFN γ)在感染及控制病毒过程中的作用和机制。方法 基于β actin的高度保守序列设计引物 ,通过RT PCR方法获得了 β actin的部分cDNA为看家基因 ,然后构建DuIFN γ靶片段的竞争性内参照 ,建立检测DuIFN γ基因转录的半定量竞争性RT PCR方法。结果 建立了检测DuIFN γ基因转录的半定量竞争性RT PCR方法 ,并检测了在PHA刺激下鸭外周血单核细胞IFN γmRNA动态变化 ,结果表明PHA刺激 2 4～ 36h ,DuIFN γmRNA转录量最高。以此方法对用DHBVpreSDNA或与IFN γDNA共免疫DHBV感染鸭进行了测定 ,结果表明IFN γ表达质粒作为DNA免疫佐剂 ,可以增强宿主IFN γ的应答。结论 IFN γ表达质粒为DNA疫苗的有效佐剂。DuIFN γ基因转录的半定量竞争性RT PCR方法为研究鸭IFN γ在DHBV感染及清除中的作用和机制奠定了基础。

Objective IFN-γ is a pleiotropic cytokine with potent immunomodulatory effects and antiviral activity. To study the mechanism of IFN-γ clearing duck hepatitis B virus (DHBV) in ducks, it is essential to establish a method to quantify expression of DuIFN-γ in immune response. In the present study,a semi-quantitative competitive RT-PCR was developed to quantify expression of duck IFN-γ(DuIFN-γ) mRNA by PBMCs. Methods Based on β-actin consensus sequence, fishing the β-actin gene as house keeping gene from duck PBMC by RT-PCR. A competitive internal control was constructed and the competitive RT-PCR system could be used to quantify the transcription of DuIFN-γ mRNA. Results After duck PBMCs were stimulated in vitro with PHA, the peak of DuIFN-γ expression was at 24-36h. Then RT-PCR method was applied to detect DuIFN-γ mRNA transcription by PBMCs from DHBV infected ducks immunized with DuIFN-γ plasmid plus DNA vaccine or DNA vaccine alone. Results showed that expression of DuIFN-γ in ducks co-immunized with DuIFN-γ plasmid were higher than other groups immunized without DuIFN-γ plasmid as adjuvant. Conclusions The results indicated that DuIFN-γ gene could be a useful adjuvant to develop vaccines. The semi-quantitation of DuIFN-γ mRNA by competitive RT-PCR provides the basis for future study of the mechanism of IFN-γ in duck hepatitis B virus persistent infection.