ABSTRACT
Objective To construct a novel prokaryotic expression system, in which cross-reacting material 197 (CRM197) can be expressed in a soluble form in Escherichia coli(E. coli) cytoplasm and purified simply by one-step Ni-NTA affinity purification. Methods The CRM197 coding sequence was cloned into the prokaryotic expres-sion vector pET-32a(+) as an fusion protein with Trx tag,the HRV3C(human rhinovirus 3C) protease recognition sequence and 6 histidine sequence were added to the N-terminal of CRM197.HRV3C protease gene was cloned into another prokaryotic expression plasmid pGArasd. Both plasmids were co-transformed into E. coli Origami B (DE3) and induced mildly at 15℃. CRM197 recombinant protein was purified by Ni-NTA affinity matrix. Results The free soluble His-tagged CRM197 protein was released by cleavage of the accompanying expressed HRV3C protease after the CRM197 fusion protein was expressed. After one-step affinity purification recombinant CRM197 protein with a purity of almost 95% was obtained. Outcoming of the final preparation incubated with DNA indicated the pu-rified CRM197 recombinant protein has deoxyribonuclease activity. Conclusions By constructing a novel double-plasmid auto-cleavage prokaryotic expression system in this study, the production process of obtaining soluble CRM197 recombinant protein in E. coli has been simplified,with expression and purification efficiency improved and the production cost reduced.
ABSTRACT
Objective To study effects of secreted cytotoxic T-lymphocyte-associated protein-4(CTLA-4)fusion Plasmodium falciparum DNA vaccine combined with granulocyte-macrophage colony stimulating factor(GM-CSF) on humoral and cellular immune responses in mice.Methods The malaria antigen coding sequence fused with CTLA-4 extracellular region of mouse was constructed as eukaryotic secretory expression vector VR 1012-sES312-CTLA,recombinant protein in culture of transfected HEK 293 cells was detected by Western blot.Balb/c mice were co-administrated with VR1012-sES312-CTLA and GM-CSF expression vector.After immunization specific antibody IgG titers and cytokines IFN-γand IL-4 expression levels were evaluated by ELISA and ELISPOT respectively. Results The introduction of CTLA-4 into malaria DNA vaccine system and application of GM-CSF adjuvant signifi-cantly enhanced the specific immune response to the vaccine.Antibody titers in VR1012-sES312-CTLA and GM-CSF co-immunized mice showed a 190-fold increase compared with the simple designed VR1012-ES312 immunization(P<0.001).Conclusions Humoral and cellular immunity induced by malaria DNA vaccine are significantly en -hanced by both dendritic cell-targeting modification and the introduction of GM-CSF molecular adjuvant into the im-mune system.This result provides a new idea for effectively raising the immune response to malaria DNA vaccine.
ABSTRACT
<p><b>BACKGROUND</b>During the blood stage of malaria infection, parasites internalize in the host red blood cells and degrade massive amounts of hemoglobin for their development. Although the morphology of the parasite's hemoglobin uptake pathway has been clearly observed, little has been known about its molecular mechanisms.</p><p><b>METHODS</b>The recombinant proteins from Plasmodium falciparum, dynamin like protein 1 (PfDYN1) and 2 (PfDYN2) GTPase domain, were expressed in E.coli and showed GTPase activity. By using a dynamin inhibitor, dynasore, we demonstrated the involvement of PfDYN1 in the hemoglobin uptake pathway.</p><p><b>RESULTS</b>The GTPase activity of the two recombinant proteins was inhibited by dynasore in vitro. Treatment of parasite cultures with 80 micromol/L dynasore at the ring and early trophozoite stage resulted in substantial inhibition of parasite growth and in an obvious decline of hemoglobin quantum. Furthermore, reduced intracellular hemozoin accumulation and decreased uptake of the FITC-dextran were also observed, together with distinctive changes in the ultrastructure of parasites after the dynasore treatment.</p><p><b>CONCLUSIONS</b>Our results show that PfDYN1 plays an important role in the hemoglobin uptake pathway of P. falciparum and suggest its possibility of being a novel target for malaria chemotherapy.</p>