[肠道病毒71型重组VP1蛋白的表达及检测肠道病毒71型感染血清学检测方法的建立]

[Expression of recombinant VP1 protein of enterovirus 71 and development of serological assay for detection of EV71 infection].

作者信息

Zhou Bo-ping, Liu Wei-long, Xie Jing-jing, Chen Xin-chun, Xu Liu-mei, Tan Yan, Liu Ying-xia, Yang Gui-lin

机构信息

The Third People's Hospital of Shenzhen, 518020, China.

出版信息

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2008 Dec;22(6):492-4.

PMID:19544656

Abstract

OBJECTIVE

To obtain a recombinant purified Enterovirus 71 VPI protein and establishment of an early, rapid and accurate serological ELISA (enzyme-linked immunosorbent assay) for detection of EV71 infection.

METHODS

VP1 gene was amplified by PCR and clonel into pET-21b (+) vector, the positive recombinant plasmid were transformed into E. coli BI21(DE3), and was induced with IPTG, the recombinant protein by SDS-PAGE and Western Blot assays. Finally, the recombinant purified VP1 protein was used as a coated antigen for detection of serum anti-IgM and IgG against EV71 by ELISA.

RESULTS

The purified VP1 was obtained, and it can be recognized by sera of patients with EV71 infection associated with hand-foot-mouth disease. The A values of anti-EV71 IgM and IgG were significantly elevated as compared to healthy objects and HFMD patients without EV71 infection (P < 0.05). The sensitivity and specificity of IgM to EV71 were 73% and 77% compared with the RT-PCR results, respectively;and those of IgG being 82% and 83%, respectively.

CONCLUSIONS

The recombinant protein VP1 was produced and purified, and it was proved to have a good antigenicity and could be used to develop a serological diagnosis kit for EV71 infection in the future.

摘要

目的

获得重组纯化的肠道病毒71型（EV71）VP1蛋白，并建立一种早期、快速、准确检测EV71感染的血清学酶联免疫吸附测定（ELISA）方法。

方法

通过PCR扩增VP1基因并克隆到pET-21b(+)载体中，将阳性重组质粒转化至大肠杆菌BL21(DE3)，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western Blot）检测重组蛋白。最后，将重组纯化的VP1蛋白作为包被抗原，通过ELISA检测血清中抗EV71的IgM和IgG。

结果

获得了纯化的VP1，其可被手足口病相关EV71感染患者的血清识别。与健康对照及无EV71感染的手足口病患者相比，抗EV71 IgM和IgG的A值显著升高（P<0.05）。与逆转录-聚合酶链反应（RT-PCR）结果相比，EV71 IgM的敏感性和特异性分别为73%和77%；IgG的敏感性和特异性分别为82%和83%。