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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 To construct an inactivated UreB / HpaA bivalent anti-Helicobacter pylori (Hp) vaccine by introducing an attenuated tri-peptide linker consisting of three glycine residues into UreB and HpaA with attenuated Salmonella typhimurium as control. The corresponding monovalent vaccine and blank vector were used to study the effect of immunoprotection on C5 7BL / 6 mice. Methods UreB / HpaA bivalent live vaccine was constructed by attenuated Salmonella typhimurium SL32 6 1 using sequence overlap extension polymerase chain reaction to amplify the fusion gene ureB / hpaA with three glycine residues flexible junctions. In vivo stability in mice. The bivalent C5 7BL / 6 mice were immunized once with the bivalent live vaccine strain. The monovalent live vaccine and blank vector were used to observe the specific antibody responses induced in vivo and the protective effect on the mice. Results The sequencing results showed that the coding sequence of three glycine residues, GGTGGAGGC, had been successfully inserted into the UreB / HpaA fusion gene. After the mice were fed with the bivalent vaccine, at least 10d remained in the spleen and terminal ileum. The bivalent vaccine induced a significant increase in serum-specific IgG1 and IgG2a levels in mice. The immune protection rate of UreB / HpaA bivalent vaccine was 77.3% (17/2 2), while that of UreB vaccine and HpaA vaccine was 50.0% (12/2) and 43.5% (10 / twenty three). Conclusion The flexible linker was introduced to optimize the construction of bivalent anti-Hp live vaccine expressing UreB and HpaA. The bivalent live vaccine of UreB / HpaA has a better immune protection effect on the second grade C5 7BL / 6 mice.