Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for diagnosis of HCV infection in Egypt

• Published in: Protein expression and purification Vol. 188; p. 105965
• Main Authors: Saleh, Eman M., Gouda, Abdullah E., Medhat, Amina M., Ahmed, Hend O., Shemis, Mohamed A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2021
• Subjects: Antibodies, Viral - blood; Antigens, Viral - biosynthesis; Antigens, Viral - genetics; Antigens, Viral - immunology; Antigens, Viral - isolation & purification; Chromatography, Ion Exchange - methods; Cloning, Molecular; Core protein; Egypt - epidemiology; ELISA; Enzyme-Linked Immunosorbent Assay; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Genotype; Guanidine - chemistry; HCV; Hepacivirus - classification; Hepacivirus - genetics; Hepacivirus - immunology; Hepatitis C - diagnosis; Hepatitis C - epidemiology; Hepatitis C - virology; Humans; Immune Sera - chemistry; Inclusion bodies; Inclusion Bodies - chemistry; Prevalence; Protein Refolding; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Refolding; Viral Core Proteins - biosynthesis; Viral Core Proteins - genetics; Viral Core Proteins - immunology; Viral Core Proteins - isolation & purification; Egypt; HCV; Core protein; Inclusion bodies; Refolding; ELISA
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2021.105965

Egypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4. This study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection. Using synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid dilution method for further purification using weak cation exchange liquid chromatography. The immunogenicity of the purified protein was tested by ELISA using 129 serum samples. The recombinant core protein was successfully expressed and purified. The results also showed that the in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the commercial ELISA kit. The sensitivity, specificity, and reproducibility of the developed assay were high and promising to be used as a screening assay for detecting HCV infection. [Display omitted] •HCV Core protein induces cellular and humoral responses, and plays a key role in the pathogenesis of HCV infection.•It potentiates the proangiogenic activity of HCC through various mechanisms to dysregulate the cell signaling pathways.•This study can be a good chance for screening new neutralizing antibodies that hinders the progress of the HCV-induced HCC.•The evaluation of the in-house assay was focused on the availability of ease, specific, and sensitive assay.•The in-house assay was tested on a group of well-characterized samples obtained from diverse regions of Egypt.•The results proved its suitability for manipulation in small laboratories.•An expected drawback of the present assay is that it may not detect HCV infection in the early infected samples.