摘要： 选取鹅黄病毒囊膜蛋白(E蛋白)基因进行克隆、表达和纯化,以获得能应用于血清学诊断和可作为亚单位疫苗的重组蛋白。根据鹅黄病毒JS804株全基因序列设计了1对引物,采用RT-PCR方法扩增出了鹅黄病毒的E基因,将其克隆到表达载体pET32a(+)中,构建了重组表达质粒pET32a-E,转化大肠杆菌BL21(DE3),并用IPTG进行诱导表达。结果表明,鹅黄病毒E基因在大肠杆菌中获得高效表达,融合蛋白分子量约为70 kDa,主要以包涵体形式存在于菌体中。Western Blot和间接免疫荧光试验发现,重组E蛋白具有较好的反应原性和良好的免疫原性。鹅黄病毒E蛋白的原核表达为进一步研究该蛋白功能、建立血清学检测方法及研制亚单位疫苗奠定了基础。
关键词:
禽黄病毒,
E蛋白,
原核表达,
抗原性
Abstract: To obtain the recombinant protein used for serologic diagnosis of and as a subunit vaccine against avian flavivirus infection,the envelope(E)gene of goose flavivirus was cloned,expressed and purified.One pair of primers was designed according to the published sequence of complete genome of the goose flavivirus strain JS804(GenBank accession number JF895923).The E gene was amplified by RT-PCR and cloned into the prokaryotic expression vector pET32a(＋) to construct the recombinant plasmid pET32a-E,which was then transformed into Escherichia coli BL21(DE3).The fusion protein was expressed after induced by IPTG and presented mainly in the form of inclusion body.The results of the Western Blot and the IFA test indicated that the expressed protein possessed strong reactinogenicity and immunogenicity.The research made a foundation for further research on the serological diagnosis and the construction and function analysis of E protein,and the E protein can serve as a potential vaccine to prevent avian flavivirus infection.
Key words:
Avian flavivirus,
E protein,
Prokaryotic expression,
Antigenicity
中图分类号:
黄欣梅, 李银, 赵冬敏, 刘宇卓, 张敬峰, 韩凯凯, 钮慧敏. 鹅黄病毒囊膜蛋白的原核表达及抗原性分析[J]. 华北农学报, 2012, 27(5): 33-37. doi: 10.3969/j.issn.1000-7091.2012.05.007.
HUANG Xin-mei, LI Yin, ZHAO Dong-min, LIU Yu-zhuo, ZHANG Jing-feng, HAN Kai-kai, NIU Hui-min. Prokaryotic Expression of the Envelope Protein of Goose Flavivirus and Antigenic Analysis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(5): 33-37. doi: 10.3969/j.issn.1000-7091.2012.05.007.