【摘要】 为获得虹鳟IFN-γ2(rt IFN-γ2)抗传染性造血器官坏死病毒(IHNV)活性的相关数据,实验根据NCBI已发表序列设计引物,提取经植物血凝素刺激后的虹鳟头肾细胞总RNA,采用RT-PCR方法扩增471 bp的该基因完整开放阅读框。将该基因重组至原核表达载体p ET32a中,并转化大肠杆菌Rosetta,进行诱导表达,SDS-PAGE结果显示,目的蛋白以包涵体形式表达,大小约为38.4 ku。重组蛋白经复性、纯化后在CHSE-214细胞上进行抗IHNV活性分析,结果显示,rt IFN-γ2在CHSE-214细胞上抗IHNV活性为6.63×106U/mg。Real-time PCR结果显示,rt IFN-γ2免疫后,虹鳟头肾、脾、肝中IRF-1、IRF-2、IFN-I、IFN-γ和Mx表达水平均显著提高,总体而言免疫后2天机体抗病毒状态弱于免疫后1天。攻毒保护实验结果显示,免疫后1天进行IHNV攻击时,鱼死亡率为40%,而免疫后2天进行IHNV攻击时,鱼死亡率达到80%。研究表明,原核表达系统制备的重组虹鳟IFN-γ2不仅具有体外抗IHNV活性,更能激发虹鳟的抗病毒状态,从而为虹鳟抵抗IHNV感染提供一定的保护力。更多还原

【Abstract】 In order to evaluate the antiviral activity against IHNV of rainbow trout IFN-γ2, the target gene(471 bp)was successfully amplified from the cultured primary head kidney leucocyte cultures which had been stimulated by PHA using the primers which were designed based on the NCBI reference sequence. Then, the gene was inserted into the p ET32 a vector and was expressed in E.coli Rosetta. The result of SDS-PAGE showed that the recombinant protein was successfully expressed in the inclusion bodies, which was about 38.4 ku. The recombinant protein could be simply purified by just washing two times. The antiviral ability of the refolding protein was evaluated in CHSE-214 cells. The activity of rt IFN-γ2 against IHNV was 6.63×106 U/mg. Based on this, the rainbow trout were injected with rt IFN-γ2. The results of real-time PCR indicated that rt IFN-γ2 was potent to induce the comparable levels of IRF-1, IRF-2, IFN-I, IFN-γ and Mx transcription in head kidney, spleen and liver. Generally, the antiviral state on day 1 post immunization was stronger than that on day 2 post immunization. In addition, its protection against IHNV was 40% and 80% when the challenge was on day 1 and day 2 post immunization, which was closely correlated with the kinetic profile of the antiviral state. In conclusion, our study here showed that the rt IFN-γ2 of rainbow trout with the activity was successfully produced in the prokaryotic expression system. Furthermore,the rt IFN-γ2 could elicit the ideal antiviral state and provide the subsequent protection against IHNV.更多还原