ABSTRACT
Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1β secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.
ABSTRACT
The relationship between the conformation of interferon-α (IFN-α) and its anti-viral activity were analyzed by circular dichroism (CD) and flow cytometry (FCM) techniques. The recombinant human IFN-α (rIFN-α2b and rIFN-α2a) were used. CD spectra from 190 nm to 240 nm indicated that two the IFN-α showed stable secondary structure at 65 degrees C, but unstable when the temperature was above 65 degrees C, and the change was irreversible. FCM data of the anti-viral activity of IFN-α indicated that the change of its secondary structures partly weakened its anti-viral activity. The rIFN-α2b and rIFN-α2a showed the same phenomenon. These data indicated that the conformation of IFN-α is one of the factors to influence its anti-viral activity and the combination of CD and FCM is a good method to analyze the relationship between the conformation of protein drugs and their biological activities in single cell level.
Subject(s)
Humans , Antiviral Agents , Chemistry , Circular Dichroism , Flow Cytometry , Interferon-alpha , Chemistry , Protein Structure, Secondary , Recombinant Proteins , ChemistryABSTRACT
Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.