新型嗜酸乳杆菌食品级载体系统的构建及其在出血性大肠埃希菌O157:H7活载体疫苗研制中的初步应用

近年来大肠埃希菌O157:H7感染频频暴发，因此迫切需要进行相关的疫苗研究。乳球菌表达系统广泛用于表达外源目的基因，是黏膜免疫抗原传递载体的理想选择，但尚无嗜酸乳杆菌构建疫苗的报道。本课题在前期成功构建高效表达O157紧密黏附素受体tir的重组嗜酸乳杆菌基础上，①用食品级筛选标记替换乳酸菌质粒pMG36e的红霉素抗性基因，构建乳酸菌食品级载体；②利用细菌营养缺陷型互补原理，制备食品级嗜酸乳杆菌宿主/载体系统；③从O157 Tir和分泌蛋白EspA两个关键黏附定植功能蛋白出发，构建O157新型双表位食品级嗜酸乳杆菌活载体疫苗候选株，体外评价其安全性、稳定性及免疫原性；④建立O157感染BALB/c小鼠模型，将疫苗候选株通过肌肉注射、滴鼻、口服三种途径免疫小鼠，测试细胞和体液免疫水平，确定最佳免疫途径，通过攻毒保护实验评价其安全性、免疫预防效果及可行性，为O157疫苗的研制探索一条新的途径。

In recent years,frequent outbreak of Escherichia co1i O157:H7 infection appearls for more urgent attention in related vaccination research. Lactic acid bacteria expression system is widely used in foreign purpose gene expression which is an ideal mucous membrane immune antigen transmission carrier, but there is no report about vaccine constructed with lactobacillus acidophilus .We constructed a recombinant Lactobacillus acidophilus that expresses high levels of O157 translocation intimin receptor(Tir),on this base,in this study we will start to work as follows: (1) Constructing the lactic acid bacteria food grade carrier with food-grade selection markers replace lactic acid bacteria pMG36e plasmid of erythromycin resistance gene; (2) Constructing nutrition defect type complementary lactobacillus acidophilus host/carrier system on the bacteria nutrition defect type complementary principle; (3) Constructing new food grade O157 bivalent lactobacillus acidophilus live vector vaccine with O157 Tir and secretory protein EspA as candidates and evaluating its safety, stability and immunogenicity in vitro; (4) Establishing O157 infected BALB/c mice model and immuning mice with the vaccine candidate strain by intramuscular injection, dripping nose, oral three ways to test cells and humoral immunity level .At last, determining the best way of immunity and evaluating its safety, immune prevention effect, function mechanism and feasibility . In general,it would explore a new way for the study of O157 vaccines.