优化构建UreB/HpaA双价幽门螺杆菌减毒活菌疫苗的免疫保护作用

Protection of mice against Helicobacter pylori infection by oral immunization with live attenuated Salmonella typhimurium expressing bivalent UreB/HpaA antigens

目的 以减毒鼠伤寒沙门菌为载体 ,通过在UreB和HpaA间引入由 3个甘氨酸残基组成的三肽柔韧接头 ,构建成UreB/HpaA双价抗幽门螺杆菌 (Hp)活疫苗 ,并对照相应单价疫苗和空白载体研究其对C5 7BL/6小鼠的免疫保护效果。方法 用序列重叠延伸聚合酶链反应扩增带 3个甘氨酸残基柔韧接头的融合基因ureB/hpaA ,进一步以减毒鼠伤寒沙门菌SL32 6 1为载体构建UreB/HpaA双价活疫苗 ,观察其在小鼠体内的稳定性。用双价活疫苗株免疫Ⅱ级C5 7BL/6小鼠 1次 ,对照单价活疫苗和空白载体观察其在体内诱导的特异抗体反应和对小鼠的免疫保护作用。结果 测序结果显示 ,3个甘氨酸残基的编码序列GGTGGAGGC已成功地插入UreB/HpaA融合基因中。双价疫苗灌喂小鼠后 ,至少能在脾脏和回肠末段存留 10d。双价疫苗在小鼠体内诱导血清特异性IgG1和IgG2a水平明显升高。UreB/HpaA双价疫苗的免疫保护率为77.3% (17/2 2 ) ,而UreB疫苗和HpaA疫苗的免疫保护率分别为5 0 .0 % (12 /2 4 )和 4 3.5 % (10 /2 3)。结论 引入柔韧接头 ,优化构建表达UreB和HpaA的双价抗Hp活疫苗。UreB/HpaA双价活疫苗对Ⅱ级C5 7BL/6小鼠有更好的免疫保护作用。

Objective Compared with corresponding monovalent vaccine and pure vaccine vector, live attenuated Salmonella typhimurium expressing bivalent antigens of Helicobacter pylori (H. pylori) , UreB/HpaA was constructed by introducing a flexible and tenacious linker into urease subunit B (UreB) and HpaA of H. pylori and its protection against H. pylori infection in mice was then investigated. Methods UreB/hpaA fusion gene was amplified form H. pylori genomic DNA using sequence overlap extension PCR (SOE-PCR). UreB/HpaA bivalent live vaccine was constructed using attenuated Salmonella typhimurium vaccine vector SL3261, and its stability in vivo was observed in C57BL/6 mice. Evaluation of protection against H. pylori infection was performed in native female C57BL/6 mice by oral immunization with a single dose of the live SL3261 vaccine strain expressing UreB/HpaA (10 8 CFU). Results Sequencing results showed that encoding sequence (GGTGGAGGC) of three glycine residues was inserted into the position between ureB and hpaA fusion gene as an adapter. Bivalent live vaccine strain could be recovered from spleen and Peyer's patches for a longer time (at least 10 days). Bivalent live vaccine induced marked elevation of the levels of serum specific IgG1 and IgG2A in mouse. The immune protection rate of UreB and HpaA bivalent live vaccine was 77.3% (17/22). However only 50.0% (12/24) of the mice immunized with SL3261 vaccine strain expressing UreB and 43.5% (10/23) of the mice immunized with SL3261 vaccine strain expressing HpaA were completely protected against H. pylori challenge. Conclusions Oral immunization of mice with bivalent UreB/HpaA live vaccine could induce protective immunity against H. pylori , and the protection rate of bivalent vaccine appears to be higher than that of monovalent vaccine.