新型鸭呼肠孤病毒σC基因原核表达及其抗原性的初步研究

摘要

应用原核表达系统表达新型鸭呼肠孤病毒σC基因，分析σC蛋白的抗原性。根据已登录的新型鸭呼肠孤病毒（NDRV） NP03株S1基因序列，针对σC基因设计并合成1对特异性引物，用RT-PCR扩增该基因，并将此片段克隆到原核表达载体 pET-30a （＋）上，将序列正确的重组质粒命名为 pET-NDRV-σC ，转化大肠杆菌BL21（DE3）感受态细胞，1.0 mmol· L -1 IPTG 37℃诱导表达。结果显示，RT-PCR扩增得到966 bp的目的片段；表达产物经 SDS-PAGE分析，获得了与预期相符的约41.3 ku的条带，重组蛋白主要以包涵体形式存在；Western blotting 分析显示，重组蛋白能与 NDRV 阳性血清发生反应。本试验首次应用原核表达系统获得NDRV σC蛋白，并证明其具有免疫反应性。%To obtain σC protein of Novel Duck Reovirus (NDRV ) from prokaryotic expression system and initial identification of its antigenicity ,theσC gene of NDRV was amplified by RT-PCR from NDRV NP03 strain using the specific primers which were designed according to the published cDNA sequence .Subsequently ,the target gene was cloned into pET-30a (+ ) prokaryotic expression vector to construct the recombinant plasmid pET-NDRV-σC , which was transformed into BL21 (DE3) and the expression of the σC gene was induced (1.0 mmol · L -1 IPTG , 37℃) .The fusion protein was identified by SDS-PAGE and purified .The antigenicity of the purified protein was characterized by Western blotting .TheσC gene was obtained with an identical sequence of 966 bp to that retrieved in GenBank .The prokaryotic expression vector for σC gene was successfully constructed as confirmed by enzyme digestion and DNA sequencing .The result of SDS-PAGE showed that the fusion protein had a relative molecular weight of 41.3 ku . Moreover , the σC protein obtained form prokaryotic expression system possessed good immunoreactivity which was showed by western blotting .Together ,our work laid foundation for further study of NDRV σC protein .