Cloning and expression and preliminary antigenicity identification for the diagnostic antigen of hepatitis E virus
10.3760/cma.j.issn.0254-6450.2015.03.018
- VernacularTitle:戊型肝炎病毒诊断抗原的克隆表达及抗原性初步鉴定
- Author:
Li WAN
1
,
2
;
Qiudong SU
;
Yao YI
;
Shengli BI
;
Chaxiang GUAN
Author Information
1. 410000长沙,中南大学湘雅医学院生理学系
2. 中国疾病预防控制中心病毒病预防控制所
- Keywords:
Hepatitis E virus;
Fusion protein N5;
Gene cloning;
Serological test
- From:Chinese Journal of Epidemiology
2015;36(3):275-279
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone,express and purify thioredoxin (named as N5),a specific diagnostic antigen of hepatitis E virus (HEV),and to initially evaluate its antigenicity and serological test performance.Methods Based on the gene sequences of HEV-ORF2 and carboxyl terminal ORF3 on GenBank,the codon was optimized by the Escherichia coli codon preference,inserted it into prokaryotic expression vector M48 following total gene synthesization,and expressed in Escherichia coli fusion protein N5 recombined with Thioredoxin (TRX).Fusion protein was purified in affinity chromatography,evaluating its antigenicity with Western blot technology,then evaluating its serological test performance using the negative and positive serum samples confirmed of HEV infection with laboratory and clinical tests.Results The recombinant plasmid expressing N5 diagnostic antigen was successfully established; high-level expression and purification to obtain soluble diagnostic antigens; Western blot results indicating fusion protein N5 can be bound specifically with the serum of HEV IgM antibody positive,showing satisfactory antigencity; using fusion protein N5 as the capture antigen to build indirect ELISA,testing 40 serum samples of HEV cases confirmed by pathogen detection and clinical diagnosis and 40 serum samples of healthy people,with the sensitivity and specificity of 95％ (38/40) and 90％ (36/40) respectively.Conclusion Recombinant plasmid expressing the HEV diagnostic antigen recombined with thioredoxin was successfully established,and soluble fusion protein N5 was obtained with high expression and strong antigenicity,promising in its future applications.
