猪流行性腹泻病毒Nsp6蛋白的分段表达及多克隆抗体制备

Expression of segmented Nsp6 protein of porcine epidemic diarrhea virus and its polyclonal antibody preparation

猪流行性腹泻病毒(PEDV)的非结构蛋白Nsp6在诱导宿主细胞自噬中起着重要作用。为进一步确定Nsp6蛋白诱导细胞自噬的关键功能域,本研究利用RT-PCR扩增技术获得了Nsp6及其3个分段基因。首先构建了重组原核表达质粒pSUMO-Nsp6,并转化至大肠杆菌Rosetta感受态中,经IPTG诱导并纯化后,所得蛋白用于小鼠免疫,制备抗Nsp6蛋白的多克隆抗体。再将Nsp6及其3个分段基因分别构建至真核表达载体pCMV-HA,并将其转染至IPEC-J2细胞进行真核表达,通过Western blot和双荧光标记法来确定蛋白表达情况。结果:Nsp6蛋白在Rosetta中成功表达,并通过免疫小鼠获得了效价为1∶10240的多克隆抗体,该抗体具有与PEDV和Nsp6蛋白特异性结合的能力;免疫荧光及Western blot结果表明,Nsp-6及其截短基因在IPEC-J2细胞中成功表达,且与制备的Nsp6多克隆抗体发生特异性结合。综上,本研究原核表达了Nsp6蛋白并制备了相应的特异性抗体,验证了Nsp6及其分段基因的在细胞中的真核表达,为进一步研究Nsp6蛋白的特性和功能奠定了基础。

The non-structural protein 6(Nsp6)of porcine epidemic diarrhea virus(PEDV)plays an important role in inducing autophagy in host cells.In order to further determine the key functional domains of Nsp6 protein in inducing autophagy,transcription-polymerase chain reaction(RT-PCR)amplification technique was utilized in this study to obtain Nsp6 and its three segment genes.Firstly,a recombinant prokaryotic expression plasmid pSUMO-Nsp6 was constructed and transformed into Escherichia coli Rosetta competent cells.After induction with IPTG and purification,the obtained protein was used for immunization of mice to generate polyclonal antibodies against Nsp6 protein.Then,Nsp6 and its three segment genes were separately constructed into a eukaryotic expression vector pCMV-HA and transfected into IPEC-J2 cells for eukaryotic expression.The expression was identified by Western blot and double fluorescence labeling.The experimental results showed that Nsp6 protein was successfully expressed in Rosetta cells,and polyclonal antibodies with a titer of 1∶10240 were obtained after immunization of mice.The antibodies exhibited specific ability to bind to PEDV and Nsp6 protein.Immunofluorescence and Western blot results showed that Nsp6 and its truncated genes were successfully expressed in IPEC-J2 cells and specifically bound to the prepared Nsp6 polyclonal antibody.In conclusion,this study successfully expressed Nsp6 protein in prokaryotes and generated corresponding specific antibodies,confirming the eukaryotic expression of Nsp6 and its segment genes in cells;which laid the foundation for further investigation of the characteristics and functions of Nsp6 protein.