distinct role of small heat shock protein 20 on hcv ns3 expression in hek-293t cell line

Background: Hepatitis C [HCV] is known as a serious blood-borne disease that infects millions of people globally. NS3 is a conserved non-structural sequence of hepatitis C virus which has a major role in activating specific CTL responses. As known, there is no effective vaccine against HCV infection, thus it is required to design a specific regimen of vaccination. Recently, the strong immunological properties of Heat shock proteins [Hsps] led to their use as immunomodulators and an antigen carrier for subunit vaccine candidates. In the current study, the role of Hsp20 was evaluated as a HCV NS3 gene carrier in mammalian cell line

Methods: At first, the recombinant plasmids of pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3 were constructed and their accuracy was confirmed by digestion and sequencing. Then, all recombinant plasmids were transfected into HEK293T cells by Lipofectamine and TurboFect gene delivery systems. Finally, the expression of proteins was assessed by fluorescent microscopy, western blotting, and flow cytometry

Results: In western blotting, the 47, 59, and 79 kDa bands were detected for pEGFPHsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3, respectively. The percentage of NS3- Hsp20-GFP protein expression was ~67% by TurboFect and ~50% by Lipofectamine indicating high potency of TurboFect delivery system. Furthermore, the expression of Hsp20 [~83%] was higher than NS3 [~58%] in the cells transfected by TurboFect using flow cytometry analysis. This result was confirmed in the expression of Hsp20-NS3 fusion [~67%] in which Hsp20 increased the delivery of HCV NS3 in vitro. The same data were obtained by Lipofectamine transfection reagent

Conclusion: Briefly, our data confirmed the role of Hsp20 as a suitable antigen carrier for DNA vaccine design

Different physical delivery systems: an important approach for delivery of biological molecules in vivo

Delivery of exogenous materials such as nucleic acids, peptides, proteins, and drugs into cells is an important strategy in modern cellular and molecular biology

Recently, the development of gene carriers for efficient gene transfer into cells has attracted a great attention. Furthermore, lack of effective drug delivery is one of the major problems of cancer chemotherapy

Many physical methods have been studied to enhance the efficiency of gene and drug delivery

These strategies help to cross the materials from membranes including needle injection, photodynamic therapy, jet injection, gene gun, electroporation, hydrodynamic injection, laser, magnetofection, and tattooing

The physical systems improve the transfer of genes from extracellular to nucleus by creating transient membrane pores using physical forces including local or rapid systemic injection, particle impact, electric pulse, ultrasound, and laser irradiation. The recent optimization techniques of transdermal patches could improve the transdermal drug delivery through the skin. Among different physical carriers, electroporation and gene gun are the most potent methods for gene transfection and drug delivery in vivo. However, the researchers have focused on enhancing their potency with the structural modifications

Regarding to numerous barriers for biomolecules delivery in cells, this review is concentrated on description and optimization of different physical delivery systems for gene or drug transfer across membrane

Evaluation of cell penetrating peptide delivery system on HPV16E7 expression in three types of cell line

Background: the poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non-viral delivery systems

Objectives: the aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non-covalent complexes with nucleic acids for delivery of pEGFP-E7 as a model antigen in vitro

Materials and Methods: DNA construct encoding HPV16 E7 [pEGFP-E7] was prepared in large scale with high purity. MPG peptide/ DNA complexes were prepared at different N/P [nitrogen/phosphate] ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide-mediated E7-GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP-positive cells by flow cytometry. Furthermore, E7-GFP expression was confirmed by western blot analysis

Results: the cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS-7 cells transfected by MPG-based nanoparticles at an N/P ratio of 15:1 showed the highest transfection efficiency and gene expression

Conclusions: the results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPGbased nanoparticles as a potent gene delivery system

Expression and purification of HCV core and core-E1E2 proteins in different bacterial strains

Background: hepatitis C virus [HCV] is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma [HCC]. Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses

Objectives: in current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems

Materials and Methods: the expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer

Results: the data showed a clear band of [tilde]573 bp for HCV core and [tilde]2238 bp for core-E1E2 genes in agarose gel. Moreover, a [tilde]21 kDa band of core protein and a [tilde]83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method

Conclusions: this study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system

live vector expressing HPV16 L1 generates an adjuvant-induced antibody response in-vivo

Background: The association between human papillomavirus [HPV] infections and cervical cancer has suggested the design of prophylactic and therapeutic vaccines against genital warts. The HPV capsid has made of two L1 and L2 coat proteins that have produced late in viral infections. Regarding to the recent studies, two commercial prophylactic vaccines have based on L1 viral like particles [VLPs] could strongly induce antibody responses, and protect human body from HPV infections. However, the use of these HPV vaccines has hindered due to their high cost and some limitations. Currently, among various vaccination strategies, live vector-based vaccines have attracted a great attention

Objectives: Herein, a non-pathogenic strain of the protozoan organism known as Leishmania tarentolae has utilized to induce potent humoral immunity in mice model

Materials and Methods: At first, cloning of HPV16 L1 gene into Leishmania expression vector has performed and confirmed by PCR and digestion with restriction enzymes. The promastigotes of Leishmania tarentolae [Ltar] have transfected with linearized DNA construct by electroporation. Protein expression has analyzed by SDS-PAGE and western blotting. Then, the immunogenicity of leishmania expressing L1 protein [Ltar-L1] has assessed in mice model

Results: Our data has indicated that subcutaneous immunization of mice with the recombinant L.tar-L1 has led to enhance the levels of IgG1 and lgG2a in comparison with control groups. Furthermore, there was no significant increase in antibody levels between two and three times of immunizations

Conclusions: The recombinant live vector was able to induce humoral immunity in mice without need of any adjuvant. However, further studies have required to increase its efficiency

Different expression systems for production of HCV structural proteins

Hepatitis C virus [HCV] is an important agent causing chronic liver infection, which often leads to liver cirrhosis and lethal hepatocellular carcinoma [HCC]. At present, there is no effective HCV vaccine for prevention of hepatic disease and the standard treatment is neither economical nor fully effective in all the patients. However, vaccination based on structural and nonstructural proteins of HCV has attracted a special interest. Different heterologous systems have been used to generate the recombinant HCV core, E1, and E2 proteins including Escherichia coli, yeast, insects and mammalian cells. Further studies showed that the amounts of HCV recombinant proteins in E. coli are more suitable and un-expensive compared to other systems. It should be considered that this system is not efficient for generation of the glycosylated proteins. Thus, the structure of proteins is an important agent of selection for expression systems. The selection of expression systems will be critical for the use of recombinant proteins as an immunogen. In this mini-review, we briefly describe different expression systems for generation of the HCV recombinant structural proteins applied in vaccine design

Induction of strong and specific humoral and T-helper 1 cellular responses by HBsAg entrapped in the Methanobrevibacter smithii archaeosomes

Application of adjuvants with microbial origins is a recently highlighted approach in the vaccinology trials. Archaeosomes are among these microbial compounds with both adjuvant and liposomal activities and features. In the present study, recombinant HBsAg encapsulated into Methanobrevibacter smithii [M. smithii] archaeosomes. Balb/c mice immunized with this compound and humoral and cytokine secretion pattern of immunized models analyzed. Frequency of IFN-gamma secreting cells in the HBsAg-containing archaeosomes group was significantly higher than HBsAg and HBsAg+C/IFA groups [p

Reverse staining method of polyacrylamide gels by imidazole-zinc salts for detection and purification of L1 capsid protein in E.coli

The human papillomavirus L1 major capsid protein [HPV L1], the basis of the current vaccines, self-assembles into virus-like particles [VLPs]. Herein, we describe the expression and purification of recombinant HPV16 L1 in E. coli system. The L1 protein was generated in a fused form using an inducible expression system. The recombinant GST-L1 fusion protein migrated as a 82 kDa protein in SDS-PAGE. The L1 proteins formed inclusion bodies which were purified by Zn+2 reverse staining of sodium dodecyl sulfate polyacrylamide gels [SDS-PAGE] as a sensitive detection method. In western blotting, the existence of a 82 kDa band for GST-L1 protein was confirmed by anti-HPV16 L1 monoclonal antibody Camvir 1. The purified protein fraction was concentrated by ultrafiltration and dialyzed against PBS. This study has implications for the development of L1 protein purification as well as chromatographic separation used by other studies. Indeed, we could present a simple method to purify L1 protein in E. coli

HPV16 E7-CT [gp96] fusion protein: molecular cloning, expression and purification of a recombinant 6xHis-tagged protein in E. coli

The development of a therapeutic vaccine against human papillomavirus [HPV] is important for the control of cervical cancer. E7 is the major transforming protein produced in cervical cancers, and therefore represents potential tumor-specific antigen that could be the target of immunotherapy for cervical cancer. Among different vaccine strategies, protein-based vaccines are capable of generating CD8+ T cell responses in vaccinated animals and humans. Recently, development of novel strategies that enhance protein vaccine potency is important for generation of effective cancer vaccines and immunotherapies. Heat shock proteins [HSPs] including Gp96 have been shown to act as potent immuno-adjuvant to enhance antigen-specific tumor immunity. Therefore, the HSP-based protein vaccines can be administered by fusing antigens to HSPs, in vitro. It has been known that the HSP fragments [e.g., N-/or C-terminal regions] as mini-chaperones are better choice for immunization. The most straightforward method to produce large amounts of recombinant protein suitable for a vaccine is to clone the gene into a prokaryotic expression vector and produce the protein in Escherichia coli. In current study, we describe cloning of the HPV16 E7 gene linked to C-terminal fragment of gp96, identification and purification of the resultant E7-CT [gp96] fusion protein for next usage as a potential vaccine candidate protein against HPV in a pre-clinical trial. The recombinant E7-CT [gp96] migrated as a 51 kDa protein in SDS-PAGE. In Western blot experiment, the existence of a 51 kDa band for rE7-CT [gp96] was confirmed by rabbit anti-His as well as mouse anti-HPV16 E7 monoclonal antibodies. The protein of interest was both in the insoluble and the soluble fraction; therefore, purification was performed under denaturating and native conditions by affinity chromatography on Ni-NTA resin using 6xHis-tag

DNA Immunization as an efficient strategy for vaccination

The field of vaccinology provides excellent promises to control different infectious and non-infectious diseases. Genetic immunization as a new tool in this area by using naked DNA has been shown to induce humoral as well as cellular immune responses with high efficiency. This demonstrates the enormous potential of this strategy for vaccination purposes. DNA vaccines have been widely used to develop vaccines against various pathogens as well as cancer, autoimmune diseases and allergy. However, despite their successful application in many pre-clinical disease models, their potency in human clinical trials has been insufficient to provide protective immunity. Several strategies have been applied to increase the potency of DNA vaccine. Among these strategies, the linkage of antigens to Heat Shock Proteins [HSPs] and the utilization of different delivery systems have been demonstrated as efficient approaches for increasing the potency of DNA vaccines. The uptake of DNA plasmids by cells upon injection is inefficient. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target Antigen-Presenting Cells [APCs] are effective in animal models. Alternatively, different regimens called prime-boost vaccination are also effective. In this regimen, naked DNA is utilized to prime the immune system and either recombinant viral vector or purified recombinant protein with proper adjuvant is used for boosting. In this review, we discuss recent advances in upgrading the efficiency of DNA vaccination in animal models