家猪Ⅲ型干扰素基因家族性质及干扰素λ,δ,ω家族的抗病毒功能研究

【作者】 陈琳琳；

【导师】 刘明秋；

【作者基本信息】 复旦大学 ， 遗传学， 2013， 硕士

【摘要】 通过BLAST方法,我们从GenBank中获得了家猪面型干扰素PoIFN-?J,-入3的标准序列,并设计特异性引物,从猪肾细胞PK-15中调取了家猪千扰素PoIFN-、l,43的cDNA序列。使用软件进行序列分析,并通过构建不同物种的IFN-?^系统发生树,揭示了各亚型么间的进化关系。为了进一步分析PoIFN-人家族的性质,构建了pcDNA3.1/myc-战s(-)B/PoIFN-、s真核表达载体,并进行表达及性质研究。首先检测了在BHK-21细胞中转录及翻译水平的表达情况,发现R)IFN-化分别具有1个及2个N端-糖基化位点。其次,鉴定了PoIFN-Xs在猪肾细胞旧RS-2及PK-15细胞中对poly I:C、FMDV、PRV的应答表达谱。此外,检测了PoIFN-Xs在扭艮S-2中诱导抗病毒基因及细胞因子的表达情况,结果显示PoIFN-?^可显著诱导MX、OAS、PKR等ISGs及细胞因子正-6、TNF-a的表达。再者,抗病毒实验表明,PoIFN-?^s在PK-15/PRV及IBRS-2/FMDV系统均具有低于P〇IFN-al2的抗病毒活性。本研究测定了化IFN-、-0),-S家族在IBRS-2/FMDV,PK-15/PRV及Marc-145/PRRSV系统的抗病毒活性,结果显不各亚型具有不同强弱的抗病毒活性。其中,P〇IFN-(〇7具有高于PoIFN-al2的抗PRV与PRRSV能力。此外,较为系统地探索了它们分别与家猪I型干扰素家族成员PoIFN-al2联合使用的抗病毒叠加效果。在曲RS-2/FMDV系统中,将1U PoIFN-历S,Ss分别与1U PoIFN-al2联合使用,抗病毒活性増加10~200倍;不同效价比的PoIFN-?^/PoIFN-al2联合使用,体现出5~500倍不等的抗病毒增强作用;Real Time PCR检测发现IFN联合处理后的把民8-2细胞中ISGs表达量协同升高。在Marc-145细胞/PRRSV/SY0608病毒系统中,将PoIFN-?^,cos,Ss分别与等体积PoIFN-od2联合使用,采用CPE观测及Real Time PCR检测方法,观察到抗病毒能力的増强,其中IFN-col,-〇)2具有最佳协同效果。在PK-15细胞/PRV病毒系统中,除POIFN-X3与等效价PoIFN-al2联用时无明显变化,其他各等效价组合均体现出抗病毒能力的増强。更多还原

【Abstract】 The sequences of porcine type III interferons-PoIFN-X,!,-X3 were obtained from GenBank by BLAST. Specific primers were designed 1:0 isolate PoIFN-人 1,-人3genes from PK-15 cells. Sequence analysis was take打 out using software. A phyloge打etic tree of the IFN-入 gene family of different species defined the evolutionary relationship of various subtypes. PoIFN-?^l,-X.3 were then cloned into pcDNA3.1/inyc-His(-)B vector and expressed in BHK-21 cells, Westem-Blot analysis indicated 化at PoIFN-人 1 and PoIFN-X3 have 1 and 2 N-glycosylation si化s,respectively. Differential expression of the PoIFN-入 subtypes was detected in IB艮S-2and PK-15 cells in response to foot-and-mouth disease viras and pseudorabies virus.PoIFN-Xs significantly induced IFN stimulate genes such as MX, OAS and PKR,as well as IL-6 and TNF-a. PoIFN-X^s possesse过 weaker antiviral activity than PoIFN-al2 in both P民V/PK-15 and IBRS-2/FMDV systeIn this study, we determined the antiviral activity of porcine IFN-X,-co,-5families in 巧RS-2/FMDV,PK-15/PRV and Marc-145/PRRSV systems. The antiviral activity varies 江 lot i打 different subtypes and PoIFN-cd? exhibits the stronger inhibitory capacity against PRV and PRRSV, which is even higher than PoIFN-al2.We also characterized the activity of combination therapies with these types of porcine interferons against different viruses. When lU PoIFN-cos and PoIFN-5s was used in combination with lU PoIFN-a 12, i*espectivelys enhanced antiviral activity of10?200 folds was observed in EBRS-^/FMDV systems. Different ratios of PoIFN-Xs/PoIFN-a 12 Ksulted in various increasing times of anti-FMDV activity ranging from 5 化 500. Enhanced induction of some ISGs was detected by 艮eal Time PCR in IBRS-2 cells after the combined treatment of inteferons. What^s more,PoIFN-⑩ 1,-访2 showed 化e stronge巧 synergetic effects against PRRSV in Marc-145 cells. In PK-15/ PRV system, all combinations exhibited synergetic activity except the combinations of PoIFN-X3 with PoIFN-al2.更多还原