表达EDA-EmIMP1的重组枯草芽孢杆菌的制备及其免疫效果评价  

Preparation and immune effect evaluation of recombinant Bacillus subtilis expressing EDA-EmIMP1

作　　者：陈志 丁可欣 林霞琳 黄潇航 黄志坚 殷光文 CHEN Zhi;DING Ke-xin;LIN Xia-lin;HUANG Xiao-hang;HUANG Zhi-jian;YIN Guang-wen(College of animal science,Fujian agriculture and forestry university/Fujian animal medicine engineering laboratory,Fuzhou 350002,China)

机构地区：[1]福建农林大学动物科学学院/福建省动物药物工程实验室

出　　处：《中国预防兽医学报》2019年第4期408-413,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金-青年项目(31502058);国家自然科学基金面上项目(31872466);福建省高校杰出青年人才培育项目(KLa16029A);福建省高校新世纪优秀人才项目(KLa17059A)

摘　　要：枯草芽孢杆菌可以作为外源蛋白的表达系统,其作为重组蛋白的递呈载体具有很大优势,而纤维连接蛋白外域A(EDA)是一种能够提高目标蛋白抗原性的佐剂。为增强巨型艾美耳球虫的免疫相关蛋白1(IMP1)的抗原性,本研究从已构建的重组质粒中扩增EDA-EmIMP1和EmIMP1基因片段,并构建重组表达质粒pHT01-EDA-EmIMP1和pHT01-EmIMP1,将获得的重组质粒转化枯草芽孢杆菌感受态细胞,制备的重组枯草芽孢杆菌鉴定正确后增殖培养。将实验鸡分为5组,分别是:EDA组(免疫pHT01-EDA-EmIMP1重组枯草芽孢杆菌)、EmIMP1组(免疫pHT01-EmIMP1重组枯草芽孢杆菌)、枯草芽孢杆菌组、空白组(PBS)和攻虫对照组。每组鸡口服相应菌液(0.3mL1010cfu/mL菌液/只)进行免疫,每隔2周免疫1次,共免疫3次,每次免疫2周后采血。三免2周后攻虫。采用ELISA方法检测各组鸡血清中EmIMP1的抗体滴度以及细胞因子IL-10和IL-4的含量;并对攻虫后各组鸡的增重、肠道病变计分以及卵囊排出量进行计算。结果显示:制备的重组枯草芽孢杆菌可以表达目的蛋白,蛋白大小为70ku。ELISA检测结果显示,EDA组的抗体效价最高,约为1∶3200;三免EDA组的IL-10和IL-4的含量均显著高于其它组(p<0.05);EDA组的卵囊排出量显著低于其它组,而枯草芽孢杆菌组和EmIMP1组的卵囊排出量也均显著低于攻虫组(p<0.05);EDA组对肠道保护效果显著优于其它经过攻虫处理的组(p<0.05),而枯草芽孢杆菌组和EmIMP1组之间无显著差异;EDA组增重显著高于攻虫组和枯草芽孢杆菌组(p<0.05)。本研究证实枯草芽孢杆菌作为EmIMP1的抗原递呈载体可以有效提高免疫原性,其中EDA佐剂组的免疫效果最好。Bacillus subtilis is an exogenous protein expression system,which has advantages as a delivery vector for expressing recombinant proteins.Fibronectin extra domain A(EDA)is an antigenic adjuvant for improving the antigenicity of related proteins.In order to enhance the antigenicity of immune-mapped protein1(IMP1)of Eimeria maxima,we ligated EDA gene and EmIMP1 gene to construct the recombinant expression plasmids of pHT01-EDA-EmIMP1 and pHT01-EmIMP1,which were transformed into B.subtilis for the recombinant protein expressions,respectively.Then,chickens were divided into five groups,including EDA group(chickens fed with B.subtilis expressing EDA-EmIMP1),EmIMP1 group(chickens fed with B.subtilis expressing EmIMP1),B.subtilis group,blank control group(chickens fed with PBS)and challenged control group(chickens fed with PBS).The recombinant bacteria or PBS were fed to the chickens every two weeks,and adminstrated for three times.The immune effect of the recombinant bacteria was evaluated by the IgG titrations and the serum IL-10 and IL-4 level by ELISA measurment,and the weight gain,lesion scores and oocyst excretion were aslo analysed after challenging.The results showed that the recombinant B.subtilis successfully express the target protein with a protein size of about 70ku.The IgG titer of chickens in EDA group was the highest,and the titer was 1∶3,200.The levels of IL-10 and IL-4 were significantly higher than that in other groups(p<0.05).The oocyst excretion of chickens in EDA group was significantly lower than that of other groups,while oocyst excretion in B.subtilis group and EmIMP1 group were also significantly lower than that of challenged control group(p<0.05).According to the lesion score,the intestinal protection for chickens in EDA group was more efficient than that of other groups except the blank control group(p<0.05).There was no significant difference between B.subtilis group and EmIMP1 group.The weight gain of EDA group was significantly higher than that of challenged control group and B.subtilis

关 键 词：枯草芽胞杆菌 胞内表达 EDA佐剂 EmIMP1 巨型艾美耳球虫 

分 类 号：S852.61[农业科学—基础兽医学]