ETEC菌毛-肠毒素多价特异性卵黄抗体的研制及体外作用效果评估  

Preparation of ETEC fimbriae-enterotoxin multivalent specific yolk antibody and evaluation of its effect in vitro

作　　者：王通[1] 李晓宇[1] 李仕彬 王丽丽[1] 李纪彬 李淑英 魏炳栋[3] 高慧君 徐永平[1,2] WANG Tong;LI Xiaoyu;LI Shibin;WANG Lili;LI Jibin;LI Shuying;WEI Bingdong;GAO Huijun;XU Yongping(School of Bioengineering,Dalian University of Technology,Dalian 116024,China;Dalian Semfeed Bio-Engineering Technology Co.,Ltd.,Dalian 116020,China;Jilin Academy of Agricutural Sciences,Changchun 130033,China;Dalian Hanwei Chicken Co.,Ltd.,Dalian 116024,China)

机构地区：[1]大连理工大学生物工程学院,辽宁大连116024 [2]大连赛姆生物工程技术有限公司,辽宁大连116020 [3]吉林省农业科学院,长春130033 [4]大连韩伟养鸡有限公司,辽宁大连116024

出　　处：《黑龙江畜牧兽医》2021年第10期120-126,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目“畜禽养殖绿色安全饲料饲养新技术研发”“猪重要疫病免疫防控新技术研究”(2018YFD0500600;2017YFD0500600)。

摘　　要：为了研制一种能够对产肠毒素大肠杆菌菌毛和毒素两大靶点有效抑制的特异性卵黄抗体产品,试验联合产肠毒素大肠杆菌K88、K99、987P、F18、F41菌毛和重组肠毒素STa-LTB-STb(SLS)制备多价抗原并免疫蛋鸡,通过水稀释-硫酸铵沉淀法结合超滤浓缩对卵黄抗体进行分离纯化,利用SDS-PAGE及ELISA测定其纯度及效价,胶体金标记法和碳氢吸附试验考察特异性卵黄抗体的吸附效果及其对菌体表面疏水性的影响,并初步探讨了特异性卵黄抗体的体外抑菌效果。结果表明:本试验所用工艺可将卵黄抗体的最终纯度提高至84.8%,多价卵黄抗体对各抗原成分效价峰值均高于或等于1∶8×10^(3),最高可达1∶3.2×10^(4),表明多价抗原具有良好的免疫原性,适于卵黄抗体的批量生产。透射电镜下观察到特异性卵黄抗体对菌体的结合吸附效果明显。LB液体培养基中,K88、K99、987P、F18和F41株ETEC的增殖均受到明显抑制。碳氢吸附试验显示,卵黄抗体的结合作用能够显著提高菌体表面的疏水性,使其倾向于从水相中脱离。说明菌毛-肠毒素多价特异性卵黄抗体能够通过与ETEC菌体高效特异性吸附结合达到显著的抑菌效果。In order to develop a specific egg yolk antibody product that can effectively inhibit the two major targets of enterotoxigenic Escherichia coli fimbriae and enterotoxin,this experiment combined enterotoxigenic Escherichia coli K88,K99,987 P,F18,F41 fimbriae and recombinant enterotoxin STa-LTB-STb(SLS)for the preparation of multivalent antigens and the immunization to laying hens.Water dilution-ammonium sulfate precipitation method was combined with ultrafiltration concentration for the separation and purification of egg yolk antibody;SDS-PAGE and ELISA were used to determine its purity and potency;colloidal gold labeling method and hydrocarbon adsorption test were used to investigate its effect on the hydrophobicity of the bacterial surface,and the adsorption to bacteria and antibacterial effect of specific egg yolk antibody was preliminary explored.The results showed that the process used in this experiment could increase the final purity of the egg yolk antibody to 84.8%,and the peak value of the multivalent egg yolk antibody to each antigen component was higher than or equal to 1∶8×10^(3),up to 1∶3.2×10^(4),which suggested that the multivalent antigen had good immunogenicity and was suitable for mass production of yolk antibody.Under the transmission electron microscope,it was observed that the binding and adsorption effect of the specific egg yolk antibody on the bacteria was obvious.In the LB liquid medium,the proliferation of the five ETEC strains K88,K99,987 P,F18 and F41 were all significantly inhibited.Hydrocarbon adsorption test showed that the binding effect of egg yolk antibody could significantly increase the hydrophobicity of the bacterial surface,making it prone to detach from the water phase.The results suggested that fimbriae-enterotoxin multivalent specific egg yolk antibody could achieve significant antibacterial effect through efficient and specific adsorption and binding with ETEC bacteria.

关 键 词：卵黄抗体 产肠毒素大肠杆菌 菌毛 肠毒素 吸附作用 抑菌 

分 类 号：S859.799[农业科学—临床兽医学]