重组多杀性巴氏杆菌毒素的猪细胞毒性及其小鼠病理模型的建立  

Porcine Cytotoxicity of Recombinant Pasteurella multocida Toxin and Establishment of Mouse(Mus musculus)Pathological Model

作　　者：李金峰 袁建林 赵勤 杜森焱[1,2,3] 伍锐 文翼平 黄小波[1,2,3] 颜其贵 曹三杰[1,2,3] LI Jin-Feng;YUAN Jian-Lin;ZHAO Qin;DU Sen-Yan;WU Rui;WEN Yi-Pi;HUANG Xiao-Bo;YAN Qi-Gui;CAO San-Jie(ResearchCenter for Swine Disease/College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;National Teaching and Experiment Center of Animal,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Science-observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technology,Ministry of Agriculture,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院/猪病研究中心,成都611130 [2]四川农业大学国家级动物类实验教学示范中心,成都611130 [3]农业部兽用药物与兽医诊断技术四川科学观测站,成都611130

出　　处：《农业生物技术学报》2023年第1期213-222,共10页Journal of Agricultural Biotechnology

基　　金：四川省“十四五”川猪重大科技专项(2021ZDZX0010);中央引导地方科技发展专项(2021ZYD0086)。

摘　　要：多杀性巴氏杆菌毒素(Pasteurella multocida toxin,PMT)作为多杀性巴氏杆菌(Pasteurella multocida)最重要的毒力因子之一,危害生猪健康,造成巨大经济损失。为鉴定重组多杀性巴氏杆菌毒素(Recombinant Pasteurella multocida toxin,r PMT)对4种猪细胞的毒性并构建其小鼠(Mus musculus)毒性病理模型。本研究构建pColdⅠ-tox A载体并可溶性表达rPMT;通过形态学观察、细胞增殖毒性(cell counting kit-8,CCK-8)检测及钙黄绿素/碘化丙啶(calcein/propidium iodide)染色探究rPMT对PK15等4种猪细胞的毒性。运用寇氏(Karber)法测定r PMT对C57BL/6J小鼠的半数致死剂量(the median lethal dose,LD50),并对rPMT攻毒后小鼠的心、肝、脾、肺、肾、小肠进行组织病理学分析。结果显示,rPMT蛋白(146kD)表达于上清;rPMT处理4种猪细胞后,PK15细胞形态变化最明显,细胞死亡较多,细胞活性显著降低(P<0.05),而IPEC细胞活性显著升高(P<0.05);r PMT对C57BL/6J小鼠的LD50为0.490 ng/g(rPMT/体重);攻毒后小鼠肠绒毛脱落,肾脏、肝脏充血严重,脾脏红髓区脾窦出血、淋巴细胞数量减少并伴有大量色素沉积。本研究鉴定发现r PMT对4种猪细胞中的PK15毒性作用最大并成功构建了rPMT对C57BL/6J小鼠的毒性病理模型,为深入研究PMT的致病机制提供了理论基础。Pasteurella multocida toxin(PMT),as one of the most important virulence factors of Pasteurella multocida,endangers the health of pigs and causes huge economic losses.To identify the toxicity of recombinant Pasteurella multocida toxin(rPMT)against 4 kinds of porcine cells,and construct the pathological model of toxicity against Mus musculus.pColdⅠ-tox A vector was constructed and rPMT was soluble expressed.Morphological observation,cell counting kit-8(CCK-8)detection and Propidium Iodide(PI)staining were used to explore the toxicity of rPMT against PK15 and other 3 kinds of porcine cells.The median lethal dose(LD50)of rPMT to C57BL/6J mice was determined by Karber method,and the histopathological analysis of heart,liver,spleen,lung,kidney and small intestine was carried out.The results showed that rPMT protein(146 kD)was successfully expressed;After treated with rPMT,PK15 cells arose obvious pathological changes,more cells died and cell activity decreased significantly(P<0.05)among the 4kinds of porcine cell,but the activity of IPEC cells increased significantly(P<0.05);The LD50of rPMT against C57BL/6J mice was 0.490 ng/g;After the challenge,the intestinal villi of mice fell off;the congestion of kidney and liver was serious;the splenic sinus,which is in the red pulp area of spleen,bled;The number of lymphocytes decreased and a large number of pigments deposited.This study finds that the toxic effect of rPMT against PK15 was the most significant among the 4 kinds of porcine cells;The cytotoxicity model of rPMT against PK15 was successfully constructed and the pathological model of toxicity aginst C57BL/6J mice was successfully constructed,which provides a theoretical basis for further study of the pathogenic mechanism of PMT.

关 键 词：重组多杀性巴氏杆菌毒素(PMT) 猪细胞 细胞毒性 小鼠病理模型 

分 类 号：S855.12[农业科学—临床兽医学]