鲤鱼TLR2过表达对下游干扰素相关免疫因子转录水平的影响

Effect of common carp TLR2 overexpression on the expression of down-stream interferon associated immune factors m RNA in epithelioma papulosum cyprini cells

目的探讨鲤鱼Toll样受体2(Cc TLR2)过表达对下游干扰素相关免疫因子转录水平的影响及其介导的抗病毒效应。方法构建过表达载体p EGFP-N1-Cc TLR2,并将p EGFP-N1-Cc TLR2和空载体p EGFP-N1同时转染鲤鱼上皮瘤(epithelioma papulosum cyprinid,EPC)细胞;采用real-time PCR检测转染后0、6、12、24、48和72 h细胞内干扰素调节因子3(interferon regulatory factor 3,IRF3)和IRF7以及干扰素刺激基因ISG15、Mx1、PKR和Viperin的m RNA转录水平;并通过鲤春病毒血症病毒(spring viraemia of carp virus,SVCV)感染实验验证TLR2的抗病毒效应。结果在EPC细胞中转染p EGFP-N1-Cc TLR2后IRF3、IRF7、ISG15、Mx1、PKR和Viperin的m RNA转录水平均出现明显上调,在转染后48 h,其转录水平分别相当于转染前的6.3倍、16.5倍、15.0倍、13.0倍、7.6倍和92.4倍,差异显著(P<0.01);与转染p EGFP-N1空载体相比,转染p EGFP-N1-Cc TLR2的EPC细胞IRF3、IRF7、ISG15、Mx1、PKR和Viperin的m RNA转录水平在转染后48 h和72 h分别上调2倍和2.2倍、1.5倍和2.4倍、3.9倍和4.7倍、3.4倍和6.7倍、5.4倍和3.7倍、6.6倍和15.6倍,差异显著(P<0.01);转染p EGFP-N1组在感染SVCV后6、12、24、48和72 h的病毒量分别是转染p EGFP-N1-Cc TLR2组的1.1倍、2.4倍、3.8倍、11.7倍、11.4倍,差异显著(P<0.01)。结论鲤鱼TLR2在EPC细胞中过表达可上调IRF3、IRF7、ISG15、Mx1、PKR和Viperin基因的m RNA转录水平,以及抑制SVCV的增殖;提示鱼类TLR2可通过激活IRF3或/和IRF7信号通路诱导I-IFN的产生,进而诱导ISG15、Mx1、PKR和Viperin等干扰素刺激基因表达抗病毒蛋白发挥抗病毒免疫效应。

Toll-like receptors （TLRs） is a class of pattern recognition receptors （PRRs） in host cells, which could recognize pathogen associated molecular patterns （PAMPs） and play an important role in the immune response to antiviral infection. In this study, to explore effect of common carp TLR2 （CcTLR2） overexpression on the expression of down-stream interferon associated immune factors mRNA in epithelioma papulosum cyprini （EPC） cells, we cloned the CcTLR2 gene and ligated it into an expression vector pEGFP-N1. Both vectors of pEGFP-N1 and pEGFP-N1-CcTLR2 were transfected into EPC cells, and then the expression of IRF3, IRF7, ISG15, Mxl, PKR and Viperin mRNA at 0, 6, 12, 24, 48 and 72 h post-transfection were determined by real-time PCR; meanwhile the spring viraemia of carp virus （SVCV） infection experiment was performed to evaluate the antivirus efficacy of TLR2. The results showed that expression of IRF3, IRF7, ISG15, Mxl, PKR and Viperin mRNA were obviously upregulated in EPC cells transfected with pEGFP-N1-CcTLR2, which were 6.3 times, 16.5 times, 15.0 times, 13.0 times, 7.6 times and 92.4 times of that in EPC cells untransfected at 48 h post-transfection （P 〈 0.01）; at 48 h and 72 h post-transfection, the expression of IRF3, IRF7, ISG15, Mxl, PKR and Viperin mRNA in EPC cells transfected with pEGFP-N1-CcTLR2 were respectively 2 times and 2.2 times, 1.5 times and 2.4 times, 3.9 times and 4.7 times, 3.4 times and 6.7 times, 5.4 times and 3.7 times, 6.6 times and 15.6 times of that in EPC cells transfected with pEGFP-N1 （P〈 0.01）; the quantity of SVCV in EPC cells transfected with pEGFP-N1 were respectively 1.1 times, 2.4 times, 3.8 times, 11.7 times and 11.4 times of that in EPC cells transfected with pEGFP-N1-CcTLR2 on 6, 12, 24, 48 and 72 h post-infection （P〈 0.01）. These results indicated that overexpression of common carp TLR2 gene in EPC cells could upregulate the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin mRNA and inhibit the reproduction of SVCV, which suggested that TLR2 of fish could activate the signaling cascade of IRF3 or/and IRF7, leading to secretion of I-IFN to induce the expression of IFN- induced genes such as ISG15, Mx1, PKR and Viperin for anti-virus effect.