Expression and bioactivity of protein transduction domain-paraoxonase 1 fusion protein / 国际药学研究杂志

ABSTRACT

Objective Using silkworm expression system to express human serum paraoxonase 1 (PON1) fusion protein with protein transduction domain P11 and to study its biological activity of fighting aginst the toxicity of dichlorvos. Methods P11-PON1 fusion gene was constructed in cloning sites of silkworm pFastBac 5B vector, the vector and was transformed to silkworm DH10BmBac competent cells. Virus particles and 5 instar silkworm was infected 96 hours after infection, parasites were collected and lyophilized crushed and preserved at-80℃. The protien was dissolved, sonicated and centrifuged before used. Supernatants were harvested. The fusion protein P11-PON1 proportion of the total protein was analyzed with SDS-PAGE electrophoresis and protein content was calculated. Mouse and zebrafish models were used to evaluate P11-PON1 fusion protein bioactivity. Each mouse was treated with 1 mg P11-PON1 fusion protein via intragastric or rectal administration. 0 and 3 hours after administration, 20 mg/kg dichlorvos were injected intraperitoneally. The status of intoxication was observed and the survival rate was scored. P11-PON1 fusion protein with concentrations of 1, 2.5, 5, 10, 20 mg/L was dissolved in zebrafish breeding water respectively. 0 and 3 hours after exposing dichlorvos were added with a final concentration of 50 mg/L. Observe their behavioral change.The survival rate of zebrafish was recorded. Results The content of P11-PON1 fusion protein was 8%of silkworm total protein. In mice experiments, P11-PON1 fusion protein by intragastric adminstration did not increase the survival of mouse. By intraperitoneal injection with dichlorvos 0h after rectal adminstration with protein,the survival rate of mouse did not significantly increase. In contrast, the mouse intraperitoneally injected with dichlorvos 3h after adminstration with protein, the survival rate increased extremely significantly (P < 0.01). In zebrafish experiments, the zebrafish exposed to dichlorvos 0 h after adminstration with protein, the survival rate was not significantly improved, while exposed to dichlorvos 3h after admindtration, the survival rate significantly increased. The survival rate of 20, 10, 5 mg/L group were 62.5%, 62.5%and 50%respectively at 24 h time point, compared to the control group. The survival rate increased extremely significantly (P < 0.01). 2.5 mg/L group was 41.7%, with the survival rate increasing significantly (P < 0.05). However, the survival rate of 1 mg/L group was 16.7%, compared to the control group. The increase had no sistatistical significance. Conclusion The PTD-containing PON1 fusion protein can be expressed in silkworm. Pretreatment with the fusion protein in mice and zebrafish decreased the toxicity of dichlorvos.