From in-silico immunogenicity verification to in vitro expression of recombinant Core-NS3 fusion protein of HCV

Abstract

BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is a serious global health burden. There is no effective vaccine against HCV and new direct acting antivirals (DAAs) are so expensive and virtually unavailable to the public. Therefore, seeking for therapeutic or prophylactic vaccines is exigent and reliever. METHODS: The secondary and tertiary structures of the recombinant Core-NS3 (rC-N) fusion protein of HCV and its B and T-cells epitopes were evaluated with bioinformatics software. Cloning and in vitro expression of rC-N were performed by pET24a(+) and E.coli BL21-DE3 expression host, respectively. The recombinant protein purifi cation was done by affi nity chromatography method and then identifi ed by Western blotting using anti-His monoclonal antibody. RESULTS: The sequences of rC-N protein consist of 1-118 amino acid parts of Core and 1095-1384 amino acids of NS3 were connected by a fl exible linker (AAY) with proteasome cleavable site. The expressed and purifi ed 46.7292 kDa rC-N protein had antigenic value up to threshold and conservancy found in this chimeric protein. Ramchandran Plot analysis represented that most residues were fallen in favourable regions. It also interacted with both type I and II major histocompatibility complex (MHC I, II) molecules. The rC-N had antigenic behaviour to create T cell responses. CONCLUSION: The results indicated that conserved rC-N protein had the ability to induce T-cell-mediated immune responses and it could be utilized as a therapeutic vaccine candidate against HCV (Tab. 3, Fig. 4, Ref. 40). Text in PDF www.elis.sk.

Preparation and characterization of PLGA Nanoparticles containing recombinant core-NS3 Fusion protein of hepatitis C virus as a nano-vaccine candidate

“…In this regard, we designed and produced a vaccine based on the conserved immune system stimulant parts of HCV, together with PLGA NPs as both an adjuvant and a delivery system. In our previous study, a rC-N fusion protein of HCV was produced as an antigen which its insilico predictions showed it can potentially induce T-cellmediated immune responses [11]. In this study, the rC-N fusion protein was connected to PLGA nanoparticles as a hydrophilic protein by a junction between the primary amin of rC-N fusion protein and the terminal carboxylic acid of PLGA NPs.…”

“…The rC-N fusion protein was prepared as described previously. Briefly, the first domain of core (amino acid residues 1-118), an AAY linker and the middle region of NS3 (aa 1095-1387) were respectively cloned into pET 24a (+) vector, expressed in Escherichia coli BL21-DE3, purified by affinity chromatography and finally analyzed by SDS-PAGE and Western Blotting using anti-6xHis tag monoclonal antibody [11]. The rC/N fusion protein and PLGA nanoparticles conjugation The biodegradable poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles were conjugated to rC/N fusion protein by a modified method used by Guo and Lee [34].…”

“…As the first step, a bipartite recombinant core-NS3 (rC-N) fusion protein, made of the first domain of core (amino acid residues 1-118) and a truncated middle region of NS3 (amino acid residues 1094-1385)were evaluated by the computational analyses . The recombinant protein was then expressed and purified successfully, as described previously [11]. The chimeric antigen in the virtual environment showed an ability to introduce CD4+ and CD8+ T-cell responses [11].However, according to the previous experiments on protein vaccines, these responses remain weak and the presence of an adjuvant ora delivery system to induce targeted and boosted immunity are expected [12].…”

“…The recombinant protein was then expressed and purified successfully, as described previously [11]. The chimeric antigen in the virtual environment showed an ability to introduce CD4+ and CD8+ T-cell responses [11].However, according to the previous experiments on protein vaccines, these responses remain weak and the presence of an adjuvant ora delivery system to induce targeted and boosted immunity are expected [12]. Nanoparticles can penetrate across biological barriers and contribute to enhanced bio-availability, controlled release of bioactive compounds, targeted delivery of vaccine antigens to the antigen-presenting cells (APCs), optimization of the immune responses and reduction in the number of boosting immunization [13,14].…”

High expression level of human epidermal growth factor (hEGF) using a well-designed fusion protein-tagged construct in E. coli

“…coli as a work horse for ongoing research has some benefi ts such as low cost, easy technique, high growth rate and great capacity to express recombinant proteins (9). Considering these general benefi ts of E. coli and overexpression of recombinant proteins in BL21 (DE3), some studies have used it to express recombinant fusion proteins (10) and antibodies (11). Different vectors have been employed to express hEGF in E. coli (12,13).…”