Brucellosis is caused by the bacterium Brucella and affects various domestic and wild species. GroEL (Heat Shock Protein 60kDa) as one of the major antigens that stimulate the immune system, increases Brucella survival. The aim of the current study was to clone and express GroEL in Escherichia coli in order to design subunit vaccine. Amplifying was performed using specific primers. The full-length open reading frame of this gene was cloned into the expression vector pET-32a(+) and expressed in BL21 (DE3). The expressed antigen was purified and the molecular weight of the recombinant protein was about 70 kDa. Sequencing results along with SDS-PAGE and Western analysis confirmed the expression of recombinant GroEL in the heterologous Escherichia coli. The results of colony polymerase chain reaction (PCR), enzyme digestion and sequencing showed that the GroEL antigen has been successfully cloned and sub-cloned into pET-32a(+). The results showed that Escherichia coli was able to express GroEL protein appropriately. This protein was expressed by induction with isopropyl β -D-thiogalactoside (IPTG) at concentration of 1 mM and it was confirmed by Ni-NTA column, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting electrophoresis. The results of this study showed that Escherichia coli can be used as an appropriate host to produce the recombinant GroEL protein. This recombinant protein may be useful to simulate immune system, to produce recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure. تفاصيل المقالة

Protective antigen (PA), a subunit of anthrax toxin from Bacillus anthracis, is known as a dominant component in subunit vaccines in protection against anthrax. In order to avoid the side effects of live attenuated and killed organisms, the use of linear neutralizing epitopes of PA is recommended in order to design recombinant vaccines. The present study is aimed at determining the dominant epitopes based on multi-parameter and multi-method analysis. The epitopes were identified by the well-known online bioinformatics server and then they were selected and compared based on the highest score and the highest repetition rate. Further analysis on predicted epitopes has been carried out by online VaxiJen 2.0 and Protein Digest server. Among the selected epitopes, those with the highest antigenicity score (>0.9 threshold) and less susceptibility to gastrointestinal tract proteases, were selected as final epitopes. Final B-cell predicted epitopes were amino acid residues 292-308, 507-521 and 706-719; residues 17-31, 315-329 and 385-400 which were determined as the best major histocompatibility complex I (MHCI) class of T-cells epitopes; in addition, residues 455-464 and 661-669 were also considered the best MCHII class of T-cells epitopes. Since random coil structure had a high probability of protein forming of antigenic epitope, the results of secondary structure analysis of the final PA epitopes have shown that all these epitopes form a 100% random coil structure. تفاصيل المقالة