Recently, the HEV vaccine that is produced in BES used Aluminum hydroxide (alum) as an adjuvant ( 12). Several adjuvants have been used in the vaccines development and amongst them aluminum hydroxide, aluminum phosphate and MF59 are widely used ( 10, 11). It is well established that adjuvants play an important role in inducing long-term immunity and enhancing the efficacy of vaccines ( 9). The truncated 112 - 578aa and 112 - 660aa of the ORF2 HEV have also been investigated in the BES ( 5, 7). The neutralizing epitope of this protein is between 457 and 607 region ( 5). The BES is an efficient insect system for HEV vaccine development because of its capacity to produce a 56 KDa protein from amino acid 112 to 607 ( 8).

So far, several efforts have been made to develop HEV vaccines through expression of different truncated ORF2 in many expression systems such as: Escherichia coli, yeast, plant, mammalian and insect cells ( 1, 5- 7). Since no cell culture system has been adopted for cultivation of HEV, the preparation of vaccine remains a great challenge. With regards to consequences and lack of specific treatment, the immunization can be an effective option for prevention of the HEV infection. All these HEV genotypes share one serotype ( 4).

Based on nucleotide sequences of ORF1, ORF2 and ORF3 regions, HEV has been classified into 5 genotypes among which genotypes 1 and 2 are reported only in humans, genotypes 3 and 4 are zoonotic infections and genotype 5 is restricted to animals. P or E2S domain is responsible for host interaction, specific immune response and genotyping determination ( 1). The capsid encompasses S, M, and P domains. Hepatitis E virus comprises three open reading frames among with Open Reading Frame 2 (ORF2) contains 660 amino acids (aa) encodes the capsid protein ( 3). The Hepatitis E virus is a non-enveloped RNA positive sense about 7.2 kilobases. Manifestations of the HEV infection in endemic regions are epidemic and sporadic hepatitis ( 4). The epidemic cases are in South and Southwest Asia, North Africa and the Middle East ( 2, 4). Based on current seroepidemiological and molecular data, the endemicity of HEV has been defined as high endemic regions comprising South and East Asia, Central Asia, Middle East and Africa. Additionally, HEV transmission has been reported via blood transfusion and food contamination derived from infected animal products ( 4). Hepatitis E virus is a water-borne infection and oral-fecal is the main route of transmission due to drinking water contamination ( 3).

The clinical manifestation of the disease varies from subclinical to severe hepatitis, which may result in liver failure with consequences of morbidity and mortality ( 1, 2). Hepatitis E virus (HEV) is a causative agent of the hepatitis E infection with a mortality rate of about 0.5% to as high as 25% in pregnant women and immunosuppression individuals especially in high endemicity regions of the world ( 1). The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 43 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The lengths of the subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 15bp, respectively. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Then the products were transformed into Escherichia coli DH5О± and retransformed in DH10Bac competent cells. The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing.

The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis.

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