ABSTRACT
The recombinant chimeric enzyme of AnsB-TTP-P277 comprising L-asparaginase, a tetanus toxin peptide spacer and P277 was expressed as a soluble protein in Escherichia coli. The purified chimeric enzyme exhibited primary activity of the native asparaginase. Prediabetic NOD mice immunized with the chimeric enzyme could induce specific antibodies against P277 and the specificity of anti-P277 antibodies was verified by Western blot assay. The study showed that displaying the P277 epitope on the surface of asparaginase could effectively overcome the weak antigenicity of the P277 epitope and evoke a strong P277-specific immune response in mice. Moreover, the concentration of blood glucose was measured by an automated analyzer.Histochemical analysis of mice pancreas tissues showed that the administration of the chimeric enzyme to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself.
ABSTRACT
Objective To investigate the effects of selective COX-2 inhibitor nimesulide on growth inhibition,apoptosis and expression of COX-2 of human esophageal carcinoma Eca-109 cell line; and analyzed the correlation with the anti-oncogene,P277~(kip1). Methods MTT assay was used to detect the proliferation of Eca-109 cell. Apoptosis and cell cycle were determined by electronic microscopy and flow cytometry. The expression of COX-2 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR),the protein expression of COX-2 and P277~(kip1) were examined by Western blot analysis. Results Nimesulide significantly inhibited the proliferation of Eca-109 cell line in a time-and dose-depenent fashion; increased the proportion of cells in the G_0/G_1 phase and induced apoptosis of the cells in a dose-dependent(manner). Meanwhile,nimesulide can down-regulated the expression of COX-2 and up-regulated the expression of P277~(kip1) protein.Conclusion Nimesulide can inhibit the proliferation of Eca-109 cells,cause G_0/G_1 phase cell cycle arrest and induce apoptosis.The mechanism is probably explained with down-regulation of the expression of COX-2 and up-regulation of P277~(kip1) expression.
ABSTRACT
AIM: To improve the prevent efficacy of peptide p277 in autoimmune diabetes. METHODS: The recombinant expression plasmid pET28-Hsp65-6×p277 was constructed by inserting 6×p277 which were amplified by PCR into the vector pET28-Hsp65. The plasmid pET28-Hsp65-6×p277 was transformed into E.coli BL21 (DE3) and the fusion protein (Hsp65-6×p277) was expressed effectively as soluble protein after inducing by lactose. The fusion protein was purified and then used to immunize 4-week old female NOD mice with three times of i.n. inoculations in the absence of adjuvants. Serum samples from the immunized mice were collected at monthly interval. The concentrations of blood glucose and antibodies were measured by automatic analyzer. RESULTS: Administration with the Hsp65-6×p277 to NOD mice could prevent the development of diabetes. CONCLUSION: The fusion protein Hsp65-6×p277 might be further developed to a vaccine against insulin-dependent diabetes mellitus.