水泡性口炎病毒N蛋白原核表达及免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of N Protein from Vesicular Stomatitis Virus

本研究旨在克隆和表达水泡性口炎病毒(vesicular stomatitis virus,VSV)特异性抗原N蛋白,进而纯化并分析其免疫原性。根据GenBank中已发表的VSV基因组N基因序列,分别合成VSV两种不同血清型的N基因,经序列对比分析后,设计合成1对特异性引物,PCR扩增获得约1 300bp的N基因片段,将目的片段亚克隆至pColdⅠ原核表达载体中,经IPTG诱导表达后,采用Ni-NTA树脂亲和层析法纯化重组N蛋白。SDS-PAGE分析表明,N基因在大肠杆菌中得到表达,蛋白大小约为50ku;Western blotting检测结果表明,该重组蛋白与VSV多克隆抗体发生特异性反应。本试验成功构建了VSV-IND和VSV-NJ的原核表达载体,实现了N蛋白在大肠杆菌中的可溶性表达,纯化后的重组蛋白具有良好的免疫原性。

This study was aimed to clone and express the specific antigen nucleoprotein（N）of vesicular stomatitis virus（VSV）,and then purify and analyze its immunogenicity.Based on published VSV genome N gene sequence in GenBank,two kinds of N genes of VSV with different serotypes were synthesized,respectively.After sequence analysis,one pair of specific primers was designed and synthesized,N gene fragment with about 1 300 bp length was amplified by PCR,and subcloned into pCold Ⅰ expression vector.The recombinant N protein was induced with IPTG and purified by Ni-NTA.The results of SDS-PAGE showed that the Ngene was successfully expressed in E.coli and the molecular weight of protein was 50ku;The results of Western blotting showed that this recombined protein specifically reacted with polyclonal antibody serum of VSV.The recombinant vector with VSV-IND and VSV-NJ were successfully constructed,and the N protein was solubly expressed in E.coli,and the purified protein demonstrated promising immunogenicity.