Expression of the VP1 protein and establishment of indirect ELISA detection method of Coxsackievirus B4
10.3760/cma.j.issn.1003-9279.2019.03.016
- VernacularTitle: 柯萨奇病毒B组4型VP1蛋白原核表达及间接ELISA检测方法的建立
- Author:
Weiwei ZHANG
1
;
Hong ZHOU
;
Juan LI
;
Zhenjie ZHANG
;
Weifeng SHI
Author Information
1. Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Shandong Universities, Taishan Medical University, Taian 271000, China
- Publication Type:Journal Article
- Keywords:
Coxsackie virus B group 4;
VP1;
Prokaryotic expression;
Indirect ELISA
- From:
Chinese Journal of Experimental and Clinical Virology
2019;33(3):303-308
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To detect antibodies to Coxsackievirus B4 (CV-B4), the indirect enzyme-linked immunosorbent assay (ELISA) method was established and optimized using the recombinant VP1 protein expressed in the prokaryote system as the envelope antigen.
Methods:The VP1 gene of CV-B4 was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). It was ligated into the expression vector pET32a(+ ) to obtain the recombinant plasmid pET32(+ )-VP1 and was then transformed into E. coli expression strain Rosetta (DE3). The recombinant VP1 protein was induced by IPTG, which was verified using SDS-PAGE electrophoresis and mass spectrometry. The establishment and optimization of the indirect ELISA reaction system was based on the purified recombinant protein mentioned above as the coating antigen.
Results:The CV-B4 VP1 gene was stably expressed in E. coli in the form of inclusion body. The optimal coating concentration of antigen was 7.5 μg per well and the optimal serum dilution was 1∶100. The threshold for determining the negative and positive result of the serum samples was the optical density value of ≥ 0.376 at 450 nm. The purified recombinant protein could be specifically recognized by CV-B4 positive serum without cross-reaction with Coxsackievirus (CV)-A, CV-B1 and CV-B5, indicating that it has good immunogenicity. However, it can cross-react with CV-B3 serum samples.
Conclusions:The indirect ELISA detection method based on the CV-B4 VP1 protein could be used in the detection of serum antibody to CV-B4 infection with good sensitivity, specificity and repeatability.
