对氧磷酶1融合蛋白的表达及其生物活性  

Expression and bioactivity of protein transduction domain-paraoxonase 1 fusion protein

作　　者：王丽兴[1,2] 张世勇[3] 吴小锋[4] 蒲韵竹[1] 陈怡君[1] 尚艳楠 查晓丹 赵恩峰[2] 赵宝全[1] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850 [2]解放军总医院妇产科,北京100853 [3]内蒙古通辽市科尔沁区第一人民医院骨一科,通辽028000 [4]浙江大学动物科学院蚕蜂科学研究所,杭州310029

出　　处：《国际药学研究杂志》2014年第3期358-362,373,共6页Journal of International Pharmaceutical Research

基　　金：国家高技术研究发展计划(863计划)资助项目(2012AA022402);北京市自然科学基金资助项目(7112109)

摘　　要：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。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＆amp;nbsp;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 ＆lt; 0.01）. In zebrafish experiments, the zebrafish exposed to dichlorvos 0 h after adminstration with protein, th

关 键 词：家蚕表达 蛋白转导域 对氧磷酶1 生物活性 

分 类 号：R965[医药卫生—药理学]