新型肠道病毒89型结构蛋白VP1的构建表达及序列分析

Sequence analysis and expression of VP1 protein of a new type of enterovirus 89

目的研究克隆表达新型肠道病毒89型VP1结构蛋白,并进行活性鉴定和序列分析。方法分离肠道病毒89型VP1结构蛋白全基因,克隆入原核表达载体PET-28构建重组质粒pH-VP1,在大肠埃希菌BL21中进行表达,检测表达产物的特异性及活性。将EV89 VP1核酸序列与Genbank数据库中的序列进行比对并建立系统进化树。结果重组质粒在1 mmol/L的IPTG 37℃诱导7 h后可诱导表达得到约33 kD的蛋白,经Western Blot实验检测证明表达的融合蛋白具有EV89抗原的活性。分离的EV89病毒其VP1区核酸序列与Genbank数据库中仅有的3株EV89序列同源性分别为86.4%、85.9%、85.6%。结论成功构建EV89重组蛋白,其表达产物具有良好的特异性及活性,可进一步用于VP1检测方法的研制。进化分析表明,分离的EV89 VP1区与Genbank数据库中其他分离株亲源性较远。

Objective To clone, express the VP1 structural protein of the new type Enterovirus 89 in E. eoli, and to identify its activity and analyze its sequence. Methods The VP1 structural protein gene of Enterovirus 89 was isolated and cloned into the prokaryotie expression vector PET-28 to construct recombinant plasmid pH-VP1, and expressed in the Escherichia coli BL21. The specificity and activity of the expression product was tested. The comparison between the nueleotide sequence of EV89 VP1 and the sequence in the Genbank was made to construct the phylogenetic tree. Results About 33 kD protein was generated after the recombinant plasmid was inducted at 37℃ by IPTG for 7 h. Through the Western Blot test, it was proved that the expressed fusion protein had the activity of the EV89 antigen. The homology between the nucleotide sequence of VP1 region of EV89 and the sequence of the only 3 EV89 in Genbank were 86.4%, 85.9%,85.6% respectively. Conclusion Suc- Cessfully construct the recombination protein of EV89, and its expression product has favorable specificity and activity, which can be further used for the research and development of VP1 testing method. The evolution analysis indicates that the isolated VP1 region of EV89 is not closely related to other isolated EV89 in the Genbank in phylogeny.