产肠毒素大肠杆菌FaeG-mSTa-LTb-STb融合表达及其包涵体口服免疫小鼠诱导的免疫应答  

Immune response of inclusion bodies from recombinant E.coli expressing Fae G-m STa-LTb-STb fusion protein by the oral route in mouse model

作　　者：赵鹏[1] 夏爽[1] 关乃瑜 谷珊珊[1] 姜艳平[1] 崔文[1] 马德星[1] 葛俊伟[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2015年第10期790-795,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31101845)

摘　　要：为研究产肠毒素大肠杆菌(ETEC)F4菌毛主要结构蛋白Fae G、STa、LTb和STb毒素融合蛋白的免疫原性,本研究采用重叠延伸PCR(SOE-PCR)扩增编码Fae G-m STa(STa突变体)-LTb-STb 4种蛋白融合基因,将其克隆于p Pro EXHTa表达载体中进行原核表达。并以表达的包涵体口服免疫BALB/c小鼠,通过检测免疫鼠的体液和细胞免疫反应,评价其免疫效果。实验结果显示,融合蛋白在大肠杆菌中以包涵体的形式高效表达,并可以被特异性抗体所识别,具有良好的反应原性。免疫小鼠后均能够产生特异性Ig G和s Ig A,并诱导产生细胞免疫应答。免疫记忆反应试验证明,免疫鼠至少在3个月内保持对特异性抗原的免疫记忆应答。本研究结果表明,Fae G-m STa-LTb-STb融合蛋白具有良好的免疫原性,口服包涵体免疫是一种可行的免疫途径,提示所制备的融合蛋白作为口服疫苗抗原具有潜在的应用价值。To evaluate the immune responses induced by inclusion bodies of the FaeG-mSTa-LTb-STb fusion protein as oral vaccine, the fusion gene of enterotoxigenic E.coli （ETEC） FaeG, mSTa （STa mutant）, LTb, and STb was amplified by SOE-PCR, and cloned into the expression vector pProEXHTa, and then transformed into E.coli BL21, The recombinant E.coli were induced with IPTG to express the fusion protein, which was detected by western blot. In addition, BALB/c mouse were used as animal model immunized via intragastric administration with inclusion bodies of the fusion protein, which were able to induce the special slgA detected in the feces, IgG and IgM in the serum by ELISA. Cellular immune responses were also detected by the spleen lymphocyte proliferation test. Our study showed the capacity of inclusion bodies to induce both systemic and mucosal responses as well as to evoke relatively-long mucosal memory （3 months）. In conclusion, inclusion bodies of FaeG-mSTa-LTb-STb fusion protein might be used as a subunit vaccine candidate to prevent F4＊ ETEC post weaning diarrhea in piglets.

关 键 词：产肠毒素大肠杆菌 断奶仔猪腹泻 多价抗原 包涵体 免疫应答 

分 类 号：S852.61[农业科学—基础兽医学]