幽门螺杆菌外膜蛋白Omp18 免疫优势表位的预测与鉴定

目的:分析预测幽门螺杆菌(Hp)外膜蛋白Omp18 的免疫优势表位,并进一步验证确定其免疫优势片段的免疫原性。方法:通过生物信息学软件DNAStar 分析预测幽门螺杆菌外膜蛋白Omp18 的潜在优势表位,并由北京赛百盛基因技术有限公司合成相应的优势片段;片段通过口服免疫的方式免疫接种雌性BALB/ c 小鼠,每周1 次共免疫4 次。末次免疫10 d后处死小鼠,取血清检测IgG、IgA 水平,取肠道灌洗液检测sIgA 水平;取小鼠脾细胞ELISA 法检测细胞上清中IFN-γ、IL-2、IL-4、IL-6 水平,并通过MTT 比色法检测脾细胞增殖情况;末次免疫10 d 后Hp 标准株攻击小鼠2 次,4 周后处死小鼠,取胃组织进行快速尿素酶实验,并计算每组的感染率。结果:ELISA 法检测各片段免疫小鼠血清IgG、IgA 以及肠道sIgA 水平均高于对照组,且差异有显著统计学意义(P<0.05);各免疫组脾细胞上清INF-γ、IL-2 水平均显著高于对照组(P<0.05),而IL-4、IL-6水平较对照组则差异无显著统计学意义(P>0.05);MTT 比色法结果显示各片段刺激后小鼠淋巴细胞均有显著增殖(P<0.05);快速尿素酶实验结果提示各片段免疫后均能显著减少小鼠胃组织中Hp 的感染(P<0.05)。结论:通过生物信息学软件分析预测的外膜蛋白Omp18 优势片段均具有较好的免疫原性,且对Hp 感染有一定的保护作用。

Prediction and identification of immunodominant epitopes in Helicobacter pylori outer membrane protein Omp18

Objective:Analysis and forecast the immunodominant epitopes of outer membrane protein Omp18 in Helicobacter pylori and further verify the immunogenicity of them.Methods: Analysis the immunodominant epitopes of outer membrane protein Omp18 by DNAStar,and than synthesis by SBS Genetech Co.,Ltd.Epitopes were immunized with female BALB/ c mice by oral immunization 1 times a week for 4 times,and the mice were sacrificed 10 d after the last immunization to detect the levels of serum IgG,IgA and intestinal perfusion sIgA.ELISA was used to detect the levels of IFN-γ,IL-2,IL-4 and IL-6 in the cell supernatant,and the proliferation of spleen cells was detected by MTT colorimetry.The mice were challenged twice with Hp standard strain 10 days after the last immunization.After 4 weeks,the mice were sacrificed to collected the gastric tissues for rapid urease test,and than calculated the infection rate of each group.Results: ELISA results indicated that immunized mice serum IgG,IgA and intestinal sIgA levels were higher than the control group(P<0.05).The levels of INF-αand IL-2 in the spleen cell supernatants of each immunization group were significantly higher than those in the control group (P<0.05),while the levels of IL-4 and IL-6 in the spleen cells were not significantly different from those in the control group (P > 0.05) .MTT colorimetric results showed that each epitopes stimulated lymphocyte proliferation was significantly(P <0.05).Rapid urease test results suggest that each epitopes after immunization could significantly reduce the infection of Hp in mouse gastric tissue(P<0.05).Conclusion: The immunodominant epitopes of outer membrane protein Omp18 in Helicobacter pylori,predicted by bioinformatics software,can provide protective effect for Hp challenge.