家猪Ⅲ型干扰素的生物化学性质与抗病毒能力研究

Biochemical and Antiviral Activity of Porcine Type Ⅲ Interferons

根据(GenBank公布的家猪Ⅲ型干扰素序列,设计特异性引物,从猪肾细胞PK-15中调取了家猪干扰素PoIFN-λ1,-λ3的cDNA序列,构建成pcDNA3.1B-/PoIFN-λs真核表达载体,并进行表达及性质研究.首先检测了在中华仓鼠肾细胞(BHK-21)中的转录及翻译水平,发现PoIFN-λ1,-λ3分别具有1个及2个N端糖基化位点.其次,鉴定了PoIFN-λs在猪肾细胞IBRS-2及PK-15中对合成双链RNA poly I:C、口蹄疫病毒(FMDV)及伪狂犬病毒(PRV)的应答表达谱.此外,检测了PoIFN-λs在IBRS-2中诱导抗病毒基因及细胞因子的表达情况,结果显示PoIFN-λs可显著诱导MX、OAS、PKR等干扰素激活基因(ISGs)及细胞因子IL-6、TNF-α的表达.抗病毒实验表明,PoIFN-λs在PK-15/PRV及IBRS-2/FMDV系统均具有低于PoIFN-α12的抗病毒活性;但在与PoIFN-α12联合使用时,抗FMDDV效果增强,Real time PCR检测发现ISGs表达量协同升高.

According to the sequences of porcine type Ⅲ interferons published in GenBank, specific primers were designed to isolate PolFN-λ1, -λ3 genes from PK-15 cells. PolFN-λ1, -λ3 were then cloned into pcDNA3.1/myc-His（-）B vector and expressed in BHK-21 cells. Western-Blot analysis indicated 1 and 2 N-glycosylation sites in PolFN-λ1 and PolFN-λ3, respectively. Differential expression of the PolFN-λ subtypes was detected in IBRS-2 and PK-15 cells in response to foot-and-mouth disease virus and pseudorabies virus. PolFN-λs significantly induced IFN stimulate genes such as MX, OAS and PKR, as well as IL-6 and TNF-α. PolFN-λs possessed weaker antiviral activity than PolFN-α12 in both PRV/PK-15 and IBRS-2/FMDV systems. However, the combination of PolFN-λs with PolFN-α12 exhibited synergistic antiviral effects against FMDV in IBRS-2 cells, while with enhanced induction of some ISGs.