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. This study is an attempt to produce a fusion protein by binding the fragment NT-gp96 in upstream of sequence of the N terminal fragment (NT300) of the NS5B gene in an expression vector.
Previous studies showed that non-structural proteins of hepatitis C virus (HCV) such as the NS5B protein could be a potent target for designing new therapeutic vaccine to stimulate the immune system. On the other hand, immunostimulatory molecules such as the N-terminal fragment of the GP96 molecule (NT-gp96) activate Toll-like receptors to secrete cytokines expressed by T cells. This study is an attempt to produce a fusion protein by binding the fragment NT-gp96 in upstream of sequence of the N terminal fragment (NT300) of the NS5B gene in an expression vector. The recombinant fusion protein was expressed in Escherichia coli (E. coli). Protein expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Results indicated that the NT-gp96-NT300 recombinant fusion protein was successfully produced in a prokaryotic (E. coli) host. The expression of the 73-kDa NT-gp96-NT300 recombinant protein was confirmed by SDS-PAGE and Western Blotting techniques. This fusion protein can be used as a novel vaccine candidate against HCV, if it is immunogenicity approved by additional immunological assays.
Hepatitis C virus (HCV) infection is associated with the development of liver disease, such as cirrhosis or hepatocellular carcinoma and a leading cause of the death and morbidity. New estimates of disease burden indicated that there are more than 180 million chronic infections worldwide, and annually three to four million people are exposed to this virus. Historically, the available HCV drug therapy is dependent on pegylated interferon (IFN) and ribavirin as first-generation combination therapy but it is associated with severe side effects (1-4). Second generation, direct-acting antivirals (DAAs) therapies have minimal side effects with a cure rate of more than 90%. Furthermore, multiple DAA therapies that target specific HCV proteins have been considered a realistic option for the future. Such drug therapies, however, have been a barrier for low-income countries because of their high costs (3, 5, 6). Therefore, there is a need for an effective therapeutic vaccine that provides the most protection against HCV. In this regard, several therapies such as recombinant vaccines, synthetic peptide vaccines, DNA vaccines, and vector vaccines have been created, but they have low efficacy in treatment and viral infection removal (7, 8, 9). Therefore, new approaches for the development of candidate vaccines have focused on attempts to increase cellular immune response by targeting conserved HCV antigens between the different HCV genotypes (10, 11, 12). Patients with HCV have a defect in their immune response mechanisms; it is believed that specialized immunostimulatory molecules can restore the immune function. Several therapeutic vaccines based on this approach, such as T-cell mediated vaccines, are undergoing Phase II clinical trials (10, 12).
HCV is a small, enveloped, positive-strand RNA virus that is classified in the Flaviviridae family. It exhibits a high degree of genetic diversity greater than HIV-targeting this unique feature of HCV has been the major subject for designing both HCV vaccines and drug therapies (7, 13). The non-structural 5 B (NS5B) protein (65 kDa, with 591 amino acids) is a RNA-dependent RNA polymerase (RdRp) that plays a key role in the replication of the HCV genome (14). Previous studies showed that non-structural proteins can be used for stimulation of the immune system, indicating that the NS5B protein could be a potent target in vaccination strategies (15). On the other hand, immunostimulatory molecules may activate Toll-like receptors to secrete cytokines expressed by T cells from patients with chronic infection (16, 17). The increase of HCV-specific immune responses in the patients with chronic infection is promising, but further research and follow-up of present clinical trials are still needed (18, 19). Therefore, in order to achieve the desired responses, one needs to combine the antigen(s) with appropriate carriers and adjuvant molecules. One of these molecules is the heat shock proteins (HSPs), a group conserved molecular chaperones that are highly effective in inducing immune responses and, therefore, can have an effect on the outcome of adaptive immune responses. Indeed, HSPs act as an immune-regulator, as well as a stimulator of the immune system (20, 21). Moreover, the HSP-antigen complex can be used in the development of vaccines against infectious disease and cancers (22, 23). Gp96 (GRP94), a 96-kDa glycoprotein, is a member of the HSP90 family. It was established that Gp96 is able to bind to receptors on professional antigen-presenting cells (APCs) and stimulate these cells (24). Moreover, the GP96-antigen complex presents the peptides to major histocompatibility complex (MHC) molecules for activation of specific CD8 and CD4 T cells, after interaction with APCs and internalization. Several studies demonstrated that the N-terminal fragment of the GP96 molecule (NT-gp96) in comparison with full-length gp96 is sufficient to bind antigens and mediate uptake of antigens into APCs; therefore, NT-gp96 could have similar effects on up-regulation of the same co-stimulatory receptors and potentially induce secretion of the same cytokines (24, 25). The source of gp96 in this study is from Xenopus laevis. The aim of this study is to clone the fragment NT-gp96 in upstream of sequence of the N-terminal fragment (NT300) of the NS5B gene in an expression vector. The designed recombinant fusion protein (rNT-gp96-NT300) was expressed and purified in E. coli (BL21) for the development of a HCV therapeutic vaccine in the future.
Construct design and cloning of fusion recombinant protein
For construction of chimeric NT-96-NT300, the NT300 fragment was first amplified by polymerase chain reaction (PCR) using pET28a expression vector (Biomatik, Ontario, Canada) containing the full coding sequence of the NS5B gene from HCV as the template and a set of primers designed as follows:
NT300 F: 5-GACGAATTCGGTACCAAGCT TTCAATGTC-3
The EcoR1 restriction site in the forward primer and Xho1 restriction sites in the reverse primer were underlined. PCR was carried out under the following conditions: 94 °C, 30 s; 61 °C, 30 s; 72 °C, 2 min for a total of 35 cycles. The PCR product of NT300 was purified by using a gel extraction kit (Fermentas, Thermo Fisher Scientific, USA), and the product was then cloned into the EcoR1/Xho1 sites of the expression vector pET28a (+), which encodes a His-tag in the C-terminal end. For amplification of the NT (gp96) region (nucleotides 16-1030), PCR was performed by using pQE30 vector (Qiagen, Germany) containing full coding sequence of gp96 as the template. Primers designed for the reaction were as follows:
NT-gp96 F: 5-CCTGGATCCGAAGATGACG
NT-gp96 R: 5-TGCGGTACCTTTGTA
The BamH1 and Kpn1 restriction sites were included in the forward and reverse primers, respectively. The PCR conditions for amplification include: running the reaction for 30 s at 94 °C; 35 s at 62 °C; 105 s at 72 °C, for a total of 35 cycles. After confirming the presence of the amplified product on a 1% agarose gel, the NT-gp96 fragment was cut and purified using a gel extraction kit (Fermentas, Thermo Fisher Scientific, USA). To generate pET28a (NT-gp96-NT300), the NT-gp96 fragment was digested with BamH1 and Kpn1 and then cloned into the BamH1/Kpn1 sites of the expression vector pET28a-NT300, upstream of the NT300 fragment. After that, the construct transformed into E. coli DH5a competent cells and the recombinant plasmids were extracted from transformed E. coli using a plasmid extraction kit (Fermentas, Thermo Fisher Scientific, USA).
PCR analysis using T7 promoter-specific primers and double digestion with Nco1 and Xho1 enzymes was used to confirm the proper insertion of the gene in the recombinant plasmid. The sequence of recombinant constructs was checked by DNA sequence analysis (Bioneer, Korea).
Expression and purification of the recombinant NT (GP96) protein-NT300
The construct, pET28a-NT (GP96)-NT300, which contains a Polyhistidine-tag (His-tag) at the C-terminus of the NT300 region, was used to transform E. coli BL21 (DE3) competent cells.
The clones were checked by PCR amplification, restriction analysis, and DNA sequencing. Expression of recombinant vector of pET28a-NT GP96-NT300 was carried out. According to the protocol, a single transformed colony of recombinant E. coli (DE3) cells was inoculated in 10 mL Luria Broth (LB) media containing 100 µg/mL kanamycin (Sigma, Germany) and grown overnight under aerobic conditions at 37 °C. The next day, 1 mL of overnight culture was inoculated to 50 mL LB broth and the cells were grown at 37 °C under aerobic conditions, with constant shaking (200 rpm). When the optical density (OD at 600 nm) of the culture reached 0.6 (OD=0.6), a final concentration of 1 mM isopropylthiogalactoside (IPTG) (Fermentas, USA) was added to the culture medium, then after induction, the culture was allowed to grow at 37 °C for an additional 6 h. After this incubation time, the cell pellet was harvested by centrifugation (4000 rpm for 15 min at 4 °C) and was stored at -20 °C until the protein purification step.
Purification of the recombinant fusion protein under denaturing condition was performed using Ni-NTA Agarose (QIAGEN, Germany) according to the manufacturer’s recommendations. Purity and identity of the recombinant protein was confirmed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.
SDS-PAGE and Western blot analysis
SDS-PAGE was used to analyze the proper expression of the NT-gp96-NT300 fusion protein. Briefly, 20 µL of each of the purified fractions were suspended in 20 µL of loading buffer, heated for 5 min at 100 °C. Then, 20 µL of each sample was used for loading on 12.5% SDS-PAGE gel.
Western blot analysis using horseradish peroxidase (HRP) conjugated anti-polyHis antibody (Sigma, USA) with an appropriate of (1:3000) dilution was performed. First, the samples were separated by SDS-PAGE. Then, the resolved samples on the gel were transferred into a nitrocellulose membrane (Millipore, Biomanufacturing and Life Science Research) using mini Trans-blot Cell (Bio-Rad, USA). Transfer was done at 4 °C at 25 Volt (V) for overnight using mini Trans-Blot Cell (Bio-Rad, USA). To prevent non-specific binding, the membrane was blocked with skimmed milk (blocking solution) in phosphate buffered saline tween-20 (PBST). To remove the excess of antibody, the membrane was washed with PBST for three times, 15 min per wash. The membrane was incubated at room temperature with conjugated anti-polyHis antibody for 2 h, under shaking condition. The horseradish peroxidase color development reagent, DAB (3, 3’-diaminobenzidine) solution (0.05%) with 30 µL hydrogen peroxidase (30%) as an enzyme substrate was used for detecting bounds binding to nitrocellulose membrane. This substrate develops a brown, insoluble product on the membrane surface after exposure to horseradish peroxidase conjugated antibodies.
Cloning and construction of rNTgp96-NT 300
The authors generated a fusion protein consisting of the NT-gp96 gene linked to the N-terminal end of the NS5B gene (NT300) by using the restriction enzyme method. The construct consists of NT-gp96 fragment, NT-300 fragment, and poly-his tag as shown in Figure 1. Results of the final PCR product and double digestion by the restriction enzymes (BamH1/Xho1) are shown in Figure 2 and Figure 3, respectively.
Figure 1. Schematic diagram of recombinant fusion construct consists of NT-gp96 fragment, NT300 fragment, and poly-his tag. (All figures are courtesy of the authors.)
Figure 2. 1. DNA ladder 1kb. 2. PCR product of fusion rNTgp96-NT 300 (1914 bp). 3. Negative control (H2O).
Figure 3. 1. 1kb DNA ladder. 2. Vector digested by (BamH1/ Xho1) enzymes (1914bp band). 3. Uncut vector.
Expression and purification of the recombinant rNTgp96-NT300 protein
Under denaturing conditions and by applying Ni-NTA affinity column, the recombinant protein His-tagged NT-gp96-NT300 was successfully purified. The result of SDS-PAGE related to the recombinant fusion protein is shown in Figure 4. Western blotting technique was performed by anti-His antibody to verify the expression of the NT-gp96-NT300 recombinant protein. The existence of main band with expected size (73 kDa) corresponding to this protein in the induced bacterial lysate is shown in Figure 5.
Figure 4. 1.Protein ladder. 2. Non-induced sample. 3. Induce sample. 4. Purified protein.
HCV infection remains a major public health problem worldwide. The current gold-standard drug therapy is limited by a number of factors (26). There is, therefore, a need to develop an effective vaccine against HCV. Up to now, there are no licensed or in-use vaccines available for the prevention or treatment of chronic HCV. One of the challenges in treating chronic HCV is immune dysfunction; in order to be effective, the therapeutic vaccine against HCV must stimulate HCV-specific immune responses (26, 27). In line with this, one of the HCV therapeutic vaccine approaches is to develop recombinant protein vaccines. Unfortunately, these proteins (antigens) have poor immunogenicity in that many of these vaccines lose their potency to stimulate appropriate immune responses. However, the application of an adjuvant could induce an Ag-specific response (28, 29). According to previous studies, the NS5B protein, when compared with other HCV antigens, is an appropriate target for a protein-based vaccine. Habersetzer F and colleagues designed and produced TG4040 as a recombinant poxvirus vaccine that expresses the HCV non-structural proteins (NSPs) (NS3, NS4, and NS5B). According to their results, the candidate vaccine induced HCV-specific cellular immune responses and reduced viral load (15). Eleanor Barnes et al. conducted a Phase Ib study of a novel virally vectored therapeutic vaccine against HCV, which contained NSPs including the NS5B protein (30).
In addition, the role of gp96 or its N-terminal fragment has been confirmed as an appropriate adjuvant in other recent studies (31). For example, E. Mohit, et al. produced the E7-NT-gp96 protein in a prokaryotic expression system, and demonstrated that protein vaccination with the fused E7-NT (gp96) protein induces potent immune response and delays tumor incidence and growth compared with the E7 protein alone (25). Hong-Tao Li and colleagues expressed and purified the gp96 and its N-terminal fragment in E. coli and reported that co-administration of the N-terminal fragment of gp96 along with HBsAg increased antibody response (32). Therefore, the authors hypothesized that fusion of GP96 as an adjuvant molecule to the NS5B protein as a target antigen could substantially enhance the immunogenicity of the antigen. This hypothesis was examined by producing the recombinant NT-gp96-NT300 fusion protein that consists of the N-terminal fragment of GP96 (NT-gp96) and the N-terminal fragment of the target antigen (NS5B)/(NT300). In this study, the authors presented the successful generation and high expression of NT-gp96-NT300 fusion protein. Cloning and expression was performed in pET28a system under IPTG inducible T7 powerful promoter. The pET28a system is one the most common and suitable systems for cloning and expression of recombinant proteins. On the other hand, E. coli BL21 (DE3) is one of the most strong and versatile expression systems because of its well-known genetics, rapid cultivation, and the inexpensive cost. The accuracy of the recombinant construct containing NT-gp96-NT300 was confirmed by PCR and sequencing analysis. According to SDS-PAGE results, recombinant proteins significantly expressed in cell lysate compared with non-induce samples. In addition, the authors used C-terminal His tag in pET28a vector for detecting protein in western blotting by anti-His conjugate-antibody and purification of fusion proteins by Ni-NTA affinity column.
The NT-gp96-NT300 recombinant fusion protein was successfully amplified, cloned, and expressed in a prokaryotic system. Future immunological studies are needed to examine NT-gp96-NT300 as a novel vaccine candidate against HCV.
The present article was extracted from the PhD thesis written by Amir Atapour, whose research was financially supported by Shiraz University of Medical Sciences, grant number 93-7369.
The authors declare that they have no conflict of interest.
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Article submitted: July 12, 2017.
Article accepted: August 8, 2017.
Volume 30, Number 10
When referring to this article, please cite it as A. Atapour et al., “Molecular Cloning, Expression, and Purification of a Recombinant Fusion Protein (rNT-gp96-NT300)," BioPharm International 30 (10) 2017.