جستجوی مقالات مرتبط با کلیدواژه "dnaj" در نشریات گروه "پزشکی"

Single‑chain fragment variable (scFv) is one of the most commonly used antibody fragments. They offer some advantages over full‑length antibodies, including better penetration to target tissues. However, their functional production has been a challenge for manufacturers due to the potential misfolding and formation of inclusion bodies. Here we evaluated the soluble expression and purification of molecular chaperone co‑expression.

E. coli BL21(DE3) cells were co‑transformed with the mixture of plasmids pKJE7 and pET22b‑scFv by the electroporation method. First, L‑arabinose was added to induce the expression of molecular chaperones, and then IPTG was used as an inducer to start the expression of anti‑HER2 scFv. The effect of cultivation temperature and IPTG concentration on soluble expression of the protein with or without chaperones was evaluated. The soluble expressed protein was subjected to native purification using the Ni‑NTA affinity column.

SDS‑PAGE analysis confirmed the successful co‑expression of anti‑HER2‑scFv and DnaK/DnaJ/GrpE chaperones. Co‑expression with chaperones and low‑temperature cultivation synergistically improved the soluble expression of anti‑HER2 scFv. Co‑expression with chaperone also exhibited an approximately four‑fold increase in the final yield of purified soluble protein.

The combination of co‑expression with chaperones and low temperature presented in this work may be useful for the improvement of commercial production of other scFvs in E. coli as functionally bioactive and soluble form.

To prevent pneumococcal infections, especially meningitis and bacteremia, and to overcome the serotype-dependent limitation of polysaccharide-based vaccines, the development of conserved protein-based vaccines is essential. This study aimed at investigate the in-silico analysis and epitope mapping of pneumococcal DnaJ for the first time, and to design the multi-epitope based vaccines with different categories by focusing on induction of both humoral and cellular immunities.

We predicted B- and T-cell epitopes, IL-4, IL-17, IL-10, and IFN-γ inducer epitopes of DnaJ using Immunoinformatics tools. The immunogenicity and conservation score of the predicted epitopes among pneumococcal prevalent clinical serotypes, the immune simulation of DnaJ administration in mammals and potential regions involved in DnaJ-TLRs interactions were analyzed. Finally, we proposed three classes of multi-epitope DnaJ-based vaccine candidates.

This protein had 24 and 15 predicted linear B-cell and helper T-cell epitopes, respectively, with a conservation score of 86-100% among prevalent clinical pneumococcal serotypes. DnaJ also had many IL-4 and IFN-γ inducing epitopes and was considered an IL-10 and IL-17 inducer protein. The immune simulation showed induction of both humoral and cellular immunity against DnaJ. The residues at positions 274, 280, 292, 297, 300, 316-319, 333, 336-340, 358,  363-366,  and 372 were predicted to be involved in DnaJ-TLR2 and DnaJ-TLR4 interactions. Three classes of proposed DnaJ-based constructs were based on only B-cell epitopes, only helper T-cell epitopes, and multi-epitopes of B- and T-cell and IL-17 epitopes.

The results showed that although DnaJ has been reported to play an important role in cellular immunity, our results indicated the high potential of DnaJ to stimulate mucosal, humoral, and cellular immunity.