CAJ | 학술논문

目的：利用家蚕表达系统表达含蛋白转导肽P11的人血清对氧磷酶1（paraoxonase 1，PON1）融合蛋白，并对其抗敌敌畏（dichlorvos，DDVP）毒性的生物活性进行初步研究。方法 P11-PON1融合基因构建于家蚕表达载体pFastBac 5B多克隆位点，转化家蚕DH10BmBac 感受态细胞，收集病毒粒子并感染家蚕5龄幼虫，感染96 h 后，收获蛋白；SDS-PAGE 电泳分析P11-PON1融合蛋白占家蚕总蛋白比例，计算蛋白含量；利用小鼠和斑马鱼鉴定P11-PON1融合蛋白抗DDVP毒性的生物活性；每只小鼠灌胃或直肠给P11-PON1融合蛋白1mg，给药后立刻和3 h后，腹腔注射DDVP溶液20 mg/kg，观察小鼠中毒状态，计算存活率；P11-PON1融合蛋白溶于斑马鱼养殖水中，浓度分别为1、2.5、5、10和20 mg/L，给药后立刻和3 h 后加入终浓度为50 mg/L的DDVP溶液染毒，观察斑马鱼的行为变化，计算存活率。结果成功表达了P11-PON1融合蛋白，该蛋白占家蚕5龄虫总蛋白的8%；灌胃给予P11-PON1融合蛋白没有提高染毒小鼠的存活率；直肠给药后立刻染毒也没有显著提高小鼠存活率，而直肠给药3 h后再腹腔注射DDVP染毒，小鼠存活率显著提高（P<0.01）；斑马鱼给予P11-PON1融合蛋白后立即染毒DDVP，斑马鱼存活率并没有显著性提高，而给予P11-PON1融合蛋白3 h后再染毒DDVP，斑马鱼存活率显著提高，其中融合蛋白20和10 mg/L组24 h存活率均为62.5%，5 mg/L组存活率为50%，与对照组相比，差异显著提高（P <0.01）；2.5 mg/L组存活率为41.7%，与对照组相比差异有显著提高（P <0.05）。结论家蚕能够表达P11-PON1融合蛋白，该融合蛋白提前给药能降低DDVP对小鼠和斑马鱼的毒性作用。
목적：이용가잠표체계통표체함단백전도태P11적인혈청대양린매1（paraoxonase 1，PON1）융합단백，병대기항활활외（dichlorvos，DDVP）독성적생물활성진행초보연구。방법 P11-PON1융합기인구건우가잠표체재체pFastBac 5B다극륭위점，전화가잠DH10BmBac 감수태세포，수집병독입자병감염가잠5령유충，감염96 h 후，수획단백；SDS-PAGE 전영분석P11-PON1융합단백점가잠총단백비례，계산단백함량；이용소서화반마어감정P11-PON1융합단백항DDVP독성적생물활성；매지소서관위혹직장급P11-PON1융합단백1mg，급약후립각화3 h후，복강주사DDVP용액20 mg/kg，관찰소서중독상태，계산존활솔；P11-PON1융합단백용우반마어양식수중，농도분별위1、2.5、5、10화20 mg/L，급약후립각화3 h 후가입종농도위50 mg/L적DDVP용액염독，관찰반마어적행위변화，계산존활솔。결과성공표체료P11-PON1융합단백，해단백점가잠5령충총단백적8%；관위급여P11-PON1융합단백몰유제고염독소서적존활솔；직장급약후립각염독야몰유현저제고소서존활솔，이직장급약3 h후재복강주사DDVP염독，소서존활솔현저제고（P<0.01）；반마어급여P11-PON1융합단백후립즉염독DDVP，반마어존활솔병몰유현저성제고，이급여P11-PON1융합단백3 h후재염독DDVP，반마어존활솔현저제고，기중융합단백20화10 mg/L조24 h존활솔균위62.5%，5 mg/L조존활솔위50%，여대조조상비，차이현저제고（P <0.01）；2.5 mg/L조존활솔위41.7%，여대조조상비차이유현저제고（P <0.05）。결론가잠능구표체P11-PON1융합단백，해융합단백제전급약능강저DDVP대소서화반마어적독성작용。
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.