ABSTRACT
<p><b>BACKGROUND</b>Cholera toxin B subunit (CTB) was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine.</p><p><b>METHODS</b>Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ (IFN-γ) enzyme-linked immunospot (Elispot) assay was used to read out the specific T-cell immunity.</p><p><b>RESULTS</b>Chloramphenicol was essential for the efficient expression of recombinant CTB (rCTB) in pET-30a/BL21 (DE3) system and could be optimized at the concentration of 0.625 µg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group ((734 ± 240) spot forming cells/10(6) splenocytes) was higher than that induced by non-adjuvanted ((520 ± 150) spot forming cells/10(6) splenocytes), all responses against different antigens were enhanced in parallel.</p><p><b>CONCLUSION</b>CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancing the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.</p>
Subject(s)
Animals , Female , Mice , Adjuvants, Immunologic , Pharmacology , Blotting, Western , Chloramphenicol , Pharmacology , Cholera Toxin , Metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Metabolism , Mice, Inbred BALB C , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , Vaccines, DNA , Genetics , Allergy and Immunology , MetabolismABSTRACT
Induced the mouse embryonic stem(ES)cells into neural cells on silk fibroin via the improved 4-/4+ RA method to explore the effect of the silk fibroin to the ES-derived neurons' growth,adherence and differentiation.Suspended the ES cells into EBs and then transferred them to three different substrates-coated 35 mm dishes including gelatin,Bombyx mori silk fibroin(SF) and Tussah silk fibroin(TSF) to identify the adherence and proportion of ES cells-derived neurons under these three substrates.The results showed that the EBs adhered to the gelatin and TSF are faster than to the SF.The average adhesive rate on gelatin and TSF are 90.3% and 84.4% respectively,and only 38.5% on SF,all the proportion of ?-Ⅲ-Tubulin positive cells is approximately 40%.It may provide important experimental information for tissue engineering,in which ES cells-derived neuron cells and silk fibroin materials are scaffolds,and also offer a source for cell therapy research of neurodegenerative disease.