【摘要】 Viperin是一种由Ⅰ型和Ⅱ型干扰素诱导的宿主蛋白,在进化上高度保守。本研究以小鼠脑组织为原材料,通过RT-PCR扩增得到包括完整open reading frame(ORF)的1 155 bp的viperin基因序列,成功亚克隆并构建了原核表达载体pET-32a-vip,将其转化到大肠杆菌Rosetta中进行原核表达。结果表明:原核表达的重组蛋白viperin主要以包涵体的形式表达,用镍离子亲和层析法获得纯度较高的viperin蛋白,为探讨原核表达的viperin抗原特性,将纯化的viperin蛋白免疫4周龄昆明小鼠,制备得到抗viperin多克隆抗体。经Western blot和IFA检测,该抗体能与真核细胞自然表达的viperin反应,特异性良好且效价高,表明原核表达的viperin具有良好的抗原性。获得的多克隆抗体为进一步研究viperin的抗病毒生物学功能奠定了良好的基础。更多还原

【Abstract】 Viperin is a kind of host cellular protein which is induced by type Ⅰ and type Ⅱ interferon and is highly conserved in evolution.In this paper,we have cloned a full-length open reading frame(ORF) of viperin gene containing 1 155 bp from brain tissue of mouse using RT-PCR.The viperin gene was successfully subcloned and reconstructed the prokaryotic recombinant expression vector pET-32a-vip,then transformed into E.coli Rosetta(DE3).The results showed that the viperin protein was expressed in a form of inclusion bodies by prokaryotic expression system.And the viperin protein was obtained with high purity by nickel ion affinity chromatography methods.To investgate antigenicity of the prokaryotic expressed viperin,the purified viperin was used to immune the Kunming mice for preparation of polyclonal antibody.Western blot and IFA analysis demonstrated that the antiviperin polyclonal antibody can react with the natural eukaryotic viperin in BHK-21 cells,and the polyclonal antibody owned well specific and high titers.These results indicated that the prokaryotic expressed viperin has well antigenicity,and it would lay foundations for further research for antiviral biological functions of viperin.更多还原