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Cloning and expression of truncated ORF2 as a vaccine candidate against hepatitis E virus.

ABSTRACT: Hepatitis E virus infection is responsible for acute viral hepatitis and associated with high mortality and still birth in pregnant women in developing countries. We report expression of truncated forms of HEV ORF2 as potential vaccine candidates for nanoparticle-based delivery. These two truncated ORF2 proteins (54 kDa and 26 kDa) have been reported to be highly immunogenic and can be used as nanoparticle-based vaccine candidate. The bacterial expressed protein was purified by affinity chromatography and further confirmed by western blot using anti-HEV antibody. The chitosan nanoemulsion was synthesized using ultrasonic waves. The nanoparticle size was found to be 120-160 nm and the entrapment efficiency of purified truncated ORF2 proteins within these nanoparticles was 70% (26 kDa) and 59% (54 kDa). In cell cytotoxicity analysis, 100 µg/mL nanoemulsion was found suitable for cell viability in both HeLa and THP1 cell lines. Release kinetics of encapsulated proteins at physiological pH 7.4 showed 26-59% and 9.7-40% release of 26 kDa and 54 kDa protein within 1 h that gradually increased with time (48 h). Encapsulated proteins were found to be unstable at pH 1.2.

SUBMITTER: Rani D

PROVIDER: S-EPMC6139098 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Cloning and expression of truncated ORF2 as a vaccine candidate against hepatitis E virus.

Rani Dibya D Saxena Rahul R Nayak Baibaswata B Srivastava Sudha S

3 Biotech 20180915 10

Hepatitis E virus infection is responsible for acute viral hepatitis and associated with high mortality and still birth in pregnant women in developing countries. We report expression of truncated forms of HEV ORF2 as potential vaccine candidates for nanoparticle-based delivery. These two truncated ORF2 proteins (54 kDa and 26 kDa) have been reported to be highly immunogenic and can be used as nanoparticle-based vaccine candidate. The bacterial expressed protein was purified by affinity chromato ...[more]

PMID: 30237961

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Project description:BACKGROUND:Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. OBJECTIVES:The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. MATERIALS AND METHODS:A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. RESULTS:Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immuno?uorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. CONCLUSIONS:The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations.

Project description:BACKGROUND:Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. OBJECTIVES:The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. MATERIALS AND METHODS:The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-?-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS:The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37? for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. CONCLUSIONS:In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.

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Cloning and expression of truncated ORF2 as a vaccine candidate against hepatitis E virus.

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Cloning and expression of truncated ORF2 as a vaccine candidate against hepatitis E virus.

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