High-level Expression of Tetanus Toxin Fragment C in Escherichia coli

Fragment C is the C-terminal domain of the heavy chain of tetanus toxin that can promote the immune response against the lethal dose of this toxin. Therefore, this portion can be considered as a candidate vaccine against tetanus infection, which occurs by Clostridium tetani. The present study aimed to compare the expression of tetanus toxin fragment C in Escherichia coli  BL21 (DE3) pLysS cells having a high tolerance to toxins between two different expression vectors, namely pET22b and pET28a, using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. After DNA extraction from Harvard CN49205 strain of C. tetani, the gene of interest was amplified using polymerase chain reaction, and then sequenced and cloned into the expression vectors of pET22b and pET28a, transformed into competent BL21 (DE3) pLysS cells, and finally expressed using an optimized protocol. The cells were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at four different incubation temperatures (i.e., 37, 33, 30, and 25 °C) and three different incubation times (i.e., 1, 2, and 3 h). Although the SDS-PAGE and western blot analyses confirmed the expression of the recombinant fragment C (r-fragment C) ligated into both of the expression vectors, pET28a showed a higher r-fragment C expression level than the other vector (38.66 mg/L versus 32.33 mg/L,

Protective Immunity Against Homologous and Heterologous Influenza Virus Lethal Challenge by Immunization with New Recombinant Chimeric HA2-M2e Fusion Protein in BALB/C Mice

Influenza is an acute and highly contagious respiratory disease. The error prone RNA polymerase and segmented nature of the influenza A virus genome allow antigenic drift and shift, respectively. Therefore, most influenza vaccines are inefficient along time and against different viral subtypes. In this study, for the first time, protection properties of a new recombinant fusion of HA2 and M2e peptides originated from influenza virus A/Brisbane/59/2007-like (H1N1) in BALB/c mice model were investigated. After immunization of the BALB/c mice, the protection property of fusion peptide was determined by a neutralizing assay test. For further study, mice were lethal challenged by the (mouse adapted, A/PR8/34 H1N1) and heterologous (mouse adapted, A/Brisbane/10/2007 H3N2) influenza virus subtypes. Then, the lung viral titers, body weight, and survival rate of the immunized mice were monitored. The results showed that immunization by the M2e-HA2 recombinant fusion peptide provides strong protection against homologous challenge and an infirm protection against heterologous. These protections against homologous and heterologous influenza A virus challenges meant the universal nature of these recombinant peptides in an immunity manner against influenza A virus. However, more studies are needed to optimize this recombinant construction, and this experiment recommends HA2-M2e fusion peptide as a universal influenza A vaccine candidate. © Copyright 2016, Mary Ann Liebert, Inc

HA03 as an Iranian Candidate Concealed Antigen for Vaccination against Hyalomma anatolicum anatolicum: Comparative Structural and In silico Studies

In the last decades researchers had focused on developing a vaccine against tick based on protective antigen. Recombinant vaccines based on concealed antigen from Boophilus microplus have been developed in Australia and Cuba by the name of TICKGARD and GAVAC (De La Fuente and Kocan, 2006). Further studies on this antigen have shown some extent of protection against other species (De Vos et al., 2001). In Iran most important species is Hyalomma anatolicum and limited information about its control are available. This paper reports structural and polymorphic analysis of HA03 as an Iranian candidate concealed antigen of H. a. anatolicum deposited in Gen-Bank .(Aghaeipour et al. GQ228820). The comparison between this antigen and other mid gut concealed antigen that their characteristics are available in GenBank showed there are high rate of similarity between them. The HA03 amino acid sequence had a homology of around 89%, 64%, 56% with HA98, BM86, BM95 respectively. Potential of MHC class I and II binding region indicated a considerable variation between BM86 antigen and its efficiency against Iranian H. a. anatolicum. In addition, predicted major of hydrophobisity and similarity in N-glycosylation besides large amount of cystein and seven EGF like regions presented in protein structure revealed that value of HA03 as a new protective antigen and the necessity of the development, BM86 homolog of H. a. anatolicum HA03 based recombinant vaccine

The inhibition of glutamine synthetase sensitizes human sarcoma cells to l-asparaginase

Purpose: To evaluate the activity of the antitumor enzyme l-asparaginase (ASNase) on tumor cells of mesenchymal origin and the contribution of glutamine synthetase (GS) to the adaptation to the metabolic stress caused by the anti-tumor enzyme. Methods: We studied the effects of ASNase in six human sarcoma cell lines: HT1080 (fibrosarcoma); RD (rhabdomyosarcoma); SW872 (liposarcoma); HOS, SAOS-2, and U2OS (osteosarcoma) in the absence or in the presence of the GS inhibitor methionine l-sulfoximine (MSO). Results: HT1080 and SW872 cells were highly sensitive to ASNase-dependent cytotoxicity. In contrast, RD, SAOS-2, HOS, and U2OS cells exhibited only a partial growth suppression upon treatment with the anti-tumor enzyme. In these cell lines ASNase treatment was associated with increased levels of GS. When ASNase was used together with MSO, the proliferation of the poorly sensitive cell lines was completely blocked and a significant decrease in the IC50 for ASNase was observed. Moreover, when ASNase treatment was carried on in the presence of MSO, HOS and U2OS osteosarcoma cells exhibited a marked cytotoxicity, with increased apoptosis. Conclusions: In human sarcoma cells (1) GS markedly contributes to the metabolic adaptation of tumor cells to ASNase and (2) the inhibition of GS activity enhances the antiproliferative and cytotoxic effects of ASNase. The two-step interference with glutamine metabolism, obtained through the combined treatment with ASNase and MSO, may provide a novel therapeutic approach that should be further investigated in human tumors of mesenchymal origin