口服表达幽门螺杆菌抗原的重组乳酸乳球菌对小鼠免疫保护作用

发布时间：2018-04-19 03:01

本文选题：幽门螺杆菌 + 乳酸乳球菌　；参考：《郑州大学》2013年硕士论文

【摘要】：目的对本课题组前期构建好的H.pylori重组疫苗菌株进行验证、诱导、表达。通过口服灌胃免疫接种BALB/c小鼠,连续免疫一个月后,用H.pylori国际标准株11637进行攻击,检测小鼠胃内H.pylori定植量,评价不同疫苗所产生的免疫应答和免疫保护效果。方法 1.将过夜培养好的乳酸菌NZ3900/pNZ8110-ureB、NZ3900/pNZ8110-lpp20、 NZ3900/pNZ8110-omp22-hpaA、NZ3900/pNZ8149-ureB、NZ3900/pNZ8110-LysM-hpaA、NZ3900/pNZ8149-SPusp45-ureB nisin诱导表达,离心提取相关蛋白,经SDS-PAGE电泳鉴定重组菌免疫蛋白表达水平,Western-blot鉴定重组蛋白免疫反应性。 2.将过夜培养好的基因工程重组菌TB1(pMAL-c2X-ureB)、TB1(pMAL-c2X-hpaA)、TB1(pMAL-c2X-lpp20)、TB1(pMAL-c2X-omp22)IPTG诱导表达超声破碎提取上清蛋白,经SDS-PAGE验证后,应用直链淀粉亲和层析柱进行纯化,使用BCA蛋白定量试剂盒测定纯化蛋白的浓度。 3.将BALB/c小鼠随机分为9组,口服免疫后,收集一半小鼠的血清和胃液,酶联免疫吸附试验(ELISA)法检测特异性抗体含量水平。剩下小鼠用H.pylori进行灌胃攻击,2周后处死小鼠,取小鼠胃组织做尿素酶半定量实验。 4.统计学分析应用SPSS17.0进行统计分析。各组资料进行正态性检验后,采用单因素方差分析对各组之间保护率进行评价。结果 1.SDS-PAGE胶照片显示Nisin诱导重组蛋白Lpp20、UreB、Omp22-HpaA、 LysM-HpaA分别在19kd、66kd、53kd、29kd处显示杂交条带,Western-blot显示各重组蛋白均有良好的免疫原性和免疫反应性。 2.各基因工程重组菌诱导表达经直链淀粉亲和层析柱纯化后,SDS-PAGE显示在相应条带均有杂交条带。 3.BALB/c小鼠口服灌胃后,NZ3900/pNZ8110-LysM-hpaA组血清IgG含量最高,其余各重组菌株组均高于阴性对照组(P0.05)。H.pylori灌胃攻击后,快速尿素酶检法显示：各重组乳酸菌组OD450值均低于对照组,差异具有统计学意义(P0.05)。结论 1.获得了纯度较高H.pylori重组抗原UreB、Lpp20、HpaA、Omp22。 2.经口服灌胃均具有免疫反应性。 3.NZ3900/pNZ8110-LysM-hpaA所引起免疫保护效果最佳
[Abstract]:PurposeThe recombinant H.pylori vaccine strains constructed by our group were verified, induced and expressed.BALB/c mice were immunized by oral administration for one month, then attacked with H.pylori international standard strain 11637 to detect the quantity of H.pylori colonization in the stomach of mice, and to evaluate the immune response and protective effect of different vaccines.Method1.NZ3900 / pNZ8110-ureBNZ3900 / pNZ8110-lpp20, NZ3900 / pNZ8110-omp22-hpaAn NZ3900 / pNZ8149-ureBNZ3900p / pNZ8110-LysM-hpaAn NZ3900pNZ8149-SPusp45-ureB nisin was induced, and the protein expression level was identified by SDS-PAGE electrophoresis.2.The recombinant gene engineering strain TB1, pMAL-c2X-ureBHPAA, TB1, pMAL-c2X-hPAA, TB1, pMAL-c2X-lpp20, TB1, pMAL-c2X-omp22, was induced to extract the protein by ultrasonic fragmentation. The protein was purified by SDS-PAGE and purified by amylose affinity chromatography. The concentration of purified protein was determined by BCA protein quantitative assay kit.3.BALB/c mice were randomly divided into 9 groups. After oral immunization, the serum and gastric juice of half of the mice were collected and the specific antibody levels were detected by enzyme linked immunosorbent assay (Elisa).The rest of the mice were killed by H.pylori for 2 weeks, and the gastric tissues of the mice were taken for the semi-quantitative test of urease.4.Statistical analysisSPSS17.0 was used for statistical analysis.After normality test, the protection rate of each group was evaluated by single factor analysis of variance (ANOVA).Result1.SDS-PAGE gel photos showed that Nisin induced recombinant protein Lpp20UreBnOmp22-HpaA, and LysM-HpaA at 19kd ~ 66kd ~ 53kdU ~ (29) kd showed that all recombinant proteins showed good immunogenicity and immunoreactivity by Western-blot.2.After purification by amylose affinity chromatography, SDS-PAGE showed that there were hybridization bands in the corresponding bands.After oral administration of 3.BALB/c mice, the serum IgG content of NZ3900 / pNZ8110-LysM-hpaA group was the highest, and that of other recombinant strains was higher than that of negative control group (P0.05N. H. pylori). The rapid urease assay showed that the OD450 value of each recombinant lactic acid bacteria group was lower than that of the control group (P 0.05).Conclusion1.High purity H.pylori recombinant antigen UreBUP Lpp20 HPA Omp22 was obtained.2.All of them were immunoreactive by oral administration.The best effect of immune protection caused by 3.NZ3900/pNZ8110-LysM-hpaA
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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