斑石鲷irf7基因克隆及其在病毒感染下的表达

Cloning and expression of irf7 gene in spotted knifejaw (Oplegnathus punctatus) under virus infection

中文关键词: 斑石鲷  干扰素调节因子7  抗病基因  免疫反应

英文关键词: Spotted knifejaw (Oplegnathus punctatus)  Interferon regulatory factor-7  Anti-virus gene  Immune response

中文摘要:

      为探究斑石鲷(Oplegnathus punctatus)干扰素调节因子irf7在虹彩病毒(Iridovirus)感染过程中的作用机制，本研究通过PCR扩增获得了斑石鲷irf7基因CDS区序列，对其序列特征进行分析，并在组织水平和细胞水平研究该基因在病毒感染中的表达模式。结果显示，Opirf7基因CDS区全长为1332 bp，编码443个氨基酸的多肽，具有干扰素调节因子家族的保守结构域。荧光定量PCR (qRT-PCR)结果显示，Opirf7基因在健康个体的不同组织中均有表达，在肝脏中表达量最高，在皮肤和肠等免疫组织中的表达量也较高。对斑石鲷腹腔注射虹彩病毒诱导抗病毒免疫反应，在组织水平检测Opirf7对病毒感染的响应模式。与对照组(0 h)相比，Opirf7在免疫组织中的表达水平有不同程度的升高。在细胞水平，建立poly I: C感染的斑石鲷脑细胞系模型，利用qRT-PCR检测Opirf7的表达变化。结果显示，poly I: C刺激后，脑细胞系Opirf7的表达量显著升高。结果表明，Opirf7在斑石鲷抗病毒病的免疫应答过程中发挥重要作用。

英文摘要:

      Interferon regulatory factor (irf7) is an immune regulatory factor that plays an important role in the antiviral process. To explore the role of irf7 in Oplegnathus punctatus (Temminck & Schlegel, 1844) under viral infection, we cloned the coding DNA sequence (CDS) region of irf7 through PCR and analyzed the expression patterns at both tissue and cell levels. The results showed that the CDS region of Opirf7 was 1 332 bp and encoded a peptide with 443 amino acids. The predicted molecular weight was 50.5 kDa and the theoretical isoelectric point was 5.546. Protein structure analysis showed that Opirf7 has three conserved domains: the DNA binding domain (DBD), IRF-associated domain (IAD), and serine-rich domain (SRD). Amino acid similarity analysis showed that OpIRF7 had the highest similarity to the IRF7 of Lates calcarifer, which was 82.92%. The similarity of Opirf7 with the IRF7 of Larimichthys crocea, Paralichthys olivaceus, and Cynoglossus semilaevis were 81.99%, 79.95%, and 73.74%, respectively. Phylogenetic analysis showed that Opirf7 and other fish irf7 genes were clustered into one branch, and irf7s from Gallus gallus, Mus musculus, Macaca mulatta, and Homo sapiens were clustered into another. Tissues from healthy O. punctatus were collected, including the liver, spleen, kidney, head kidney, intestine, gill, skin, muscle, brain, heart, and blood. After RNA extraction and cDNA synthesis, real-time PCR (qPCR) was performed to detect the expression level of Opirf7 using the comparative CT method (2−ΔΔCT method). The results of qPCR showed that Opirf7 was expressed in different tissues of healthy individuals and its expression was highest in the liver, followed by the skin and intestines. The lowest expression was observed in the head kidney. In this study, the expression profiles of Opirf7 before and after viral infection were determined at the tissue and cell levels. For the in vivo challenge study, fish were intraperitoneally injected with spotted knifejaw iridovirus, and the expression level of Opirf7 was tested in the spleen, kidney, and liver. Compared with the control group at 0 h, the expression level of Opirf7 was 15-fold in the spleen and 3-fold in the kidney 4 days after infection, and the expression peak was at 7 days after infection. However, the expression of Opirf7 was not significantly altered in the liver. A poly I:C-infected O. punctatus brain cell model was established, and the expression profiles of Opirf7 mRNA were detected before and after infection. The expression of Opirf7 mRNA in the low and medium concentration groups (50 μg/mL and 100 μg/mL, respectively) increased by 13 to 17 times, and the expression level of Opirf7 mRNA in the high concentration group (200 μg/mL) increased by approximately 8 times. It was speculated that the high concentration of 200 μg/mL caused some damage to the cells and that the expression level in the high concentration group was lower than that in the low and medium groups. In this study, the full-length open reading frame sequence of Opirf7 was cloned and characterized for the first time. The deduced amino acid sequence displayed a structure similar to those of other vertebrates. Further functional analysis showed that Opirf7 has a significant response to viral infection at both tissue and cell levels. This study demonstrated that the Opirf7 gene might play an important role in the antiviral response of O. punctatus and provide a potential molecular marker for antivirus breeding of O. punctatus.