Li Yi Ping, Bang Dang D, Handberg Kurt J, Jorgensen Poul H, Zhang Man Fu
Department of Poultry, Fish and Fur Animals, Danish Institute for Food and Veterinary Research, DK-8200 Aarhus, Denmark.
Vet Microbiol. 2005 Oct 31;110(3-4):155-65. doi: 10.1016/j.vetmic.2005.06.014.
Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real-time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including beta-actin, 28S rRNA, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 microl) total RNA. The results showed that beta-actin, 28S rRNA, 18S rRNA and GAPDH were the most constantly expressed genes, while TBP and beta-2-microglobulin were markedly induced during the infection course. Of these constant expressed genes, 28S rRNA and 18S rRNA are highly expressed; beta-actin intermediately expressed and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2 virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection.
传染性法氏囊病病毒（IBDV）可导致鸡发病，其特征为免疫抑制和高死亡率。目前，实时逆转录聚合酶链反应（RT-PCR）已用于定量病毒特异性RNA，并更好地了解宿主对感染的反应。然而，定量实时RT-PCR需要以合适的内参进行标准化。因此，我们研究了6种鸡基因的表达模式，包括β-肌动蛋白、28S核糖体RNA（rRNA）、18S rRNA、甘油醛-3-磷酸脱氢酶（GAPDH）、TATA盒结合蛋白（TBP）和β2微球蛋白，在鸡胚（CE）细胞培养物经IBDV感染7天后的表达情况。用不同感染复数（MOI）的IBDV疫苗株Bursine-2接种CE细胞，基于从标准化（100 ng）或未标准化（10 μl）总RNA合成的cDNA，通过定量实时PCR测量基因表达。结果显示，β-肌动蛋白、28S rRNA、18S rRNA和GAPDH是表达最稳定的基因，而TBP和β2微球蛋白在感染过程中明显上调。在这些稳定表达的基因中，28S rRNA和18S rRNA高表达；β-肌动蛋白中度表达，GAPDH在CE细胞培养物中的表达水平较低。此外，β-肌动蛋白在标准化和未标准化检测中以及接种病毒剂量无关的情况下均无显著变化，而其他基因有变化。β-肌动蛋白进一步成功用作内参，以定量CE细胞培养物中Bursine-2病毒特异性RNA载量。因此，β-肌动蛋白被认为是研究IBDV感染后CE细胞中基因表达以及病毒特异性RNA载量的合适内参。
