Expression and Antigen Activity Determination of Human Thyroid Peroxidase in Silkworm and Yeast

In this study,we report the expression of human thyroid peroxidase（TPO） in silkworm larvae and Pichia pastoris GS115. Recombinant TPO is sequentially purified from the hemolymph of infected silkworm larvae and yeast using a Ni-NTA resin kit. The concentration of yield of recombinant TPO is 4.87 mg per thousand larvae and 40.83 mg per liter yeast culture. However,the recombinant TPO produced in silkworm show similar binding ability with the specific anti-TPO serum to standard human TPO purified from insect cells. The lower antigen activity indicates the TPO expressed in yeast is not suitable to be used as the coating antigen in enzyme linked immunosorbent assay（ELISA）. The cost of TPO expressed in B. mori is about 1/4 that of in insect cells,and the cost of TPO purified from silkworm for ELISA is only 1/8 that of TPO produced from Sf9 cells. It indicates the Bm NPV-silkworm expression system is a cost-effectiv e platform for producing TPO with high antigen activity.

Inhibitory Effects of Berberine on the Activation and Cell Cycle Progression of Human Peripheral Lymphocytes

The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate the effect of berberine on the activation and proliferation of lymphocytes, in particular T lymphocytes. Whole peripheral blood from healthy donors was stimulated with phytohemagglutinin （PHA） alone or phorbol dibutyrate （PDB） plus ionomycin, and the expression of CD69 and CD25 on T lymphocytes was evaluated with flow cytometry. The distribution of cell cycles and cell viability were analyzed by staining with propidium iodide （PI） and 7-aminoactinomycin D （7-AAD）, respectively. The results showed that 100 μmol/L and 50 μmol/L of berberine significantly inhibited CD69 expression on T cells stimulated with PDB plus ionomycin or PHA, whereas the effect of 25 μmol/L berberine was not significant. As the incubation time increased, the extent of inhibition decreased. Similarly, the expression of CD25 was also reduced by berberine in a dose-dependent manner over the concentration range of 25-100 μmol/L. Besides, this alkaloid could block lymphocyte cell cycle progression from G0/G1 phase to S and G2/M phase without phase specificity. Moreover, analysis following 7-AAD staining revealed that berberine had no significant cytotoxicity on lymphocytes. Taken together, berberine significantly inhibits the expression of activation antigens on T lymphocytes and also blocks the progression of cell cycles of lymphocytes, suggesting that berberine may exert immunosuppressive effect through inhibiting the activation and proliferation of T cells.