猪繁殖与呼吸综合征病毒主要结构蛋白基因半定量RT-PCR方法的建立及其在RNA干扰研究中的应用

Development and Application of Semi-quantitive RT-PCR for the Main Structural Protein Genes of Porcine Reproductive and Respiratory Syndrome Virus

为了建立以GAPDH基因为参照的半定量RT-PCR检测方法,根据PRRSV VR2332株病毒基因组和GAPDH序列设计4对特异性引物,分别扩增GP5、M、N和GAPDH基因序列,将shRNA表达质粒和GP5、M、N真核表达质粒共转染HEK293A细胞,应用该法检测了转染孔中GP5、M、N的相对表达量。同管和分管扩增法均建立了以GAPDH基因为参照的半定量RT-PCR检测方法。靶向GP5、M和N蛋白基因的shRNA表达质粒对其各自蛋白的抑制率为36%～69%,其中pSi-N3和pSi-G1抑制效果最为显著。结果表明,建立的半定量RT-PCR可以用于PRRSV主要结构蛋白基因表达水平的检测分析中。

Three pairs of primers were designed according to the sequence of PRRSV VR2332 strain to amplify GP5, M and N protein gene respectively, a semi-quantitative RT-PCR method was developed. Different shRNA expressing vectors were cotransfected into HEK293A ceils with vectors expressing structural proteins of PRRSV, and the mRNA levels of target gene were assayed by semi quantitative RT-PCR. The results showed that the mRNA levels of structural proteins were inhibited from 36% to 69 %, and pSi-N3 and pSi-G1 showed the strongest inhibition effect. It indicated that the method was suitable for relative quantitation of the main structural proteins of PRRSV.