【作者】 刘喜东；

【导师】 刘明远；

【作者基本信息】 吉林大学 ， 人兽共患疫病学， 2014， 博士

【摘要】 旋毛虫病是由摄入未煮熟含有旋毛虫包囊的肉类引起的，对人类健康依然构成了巨大的威胁。沙门氏菌携带靶基因可以有效的诱导细胞和体液免疫。口服携带靶向基因的沙门氏菌口服可以模拟旋毛虫自然感染过程，减毒沙门菌疫苗经口服进入宿主肠道被Peyer淋巴结内的吞噬细胞吞噬。这些吞噬细胞被激活后开始迁移至全身，沙门菌裂解后质粒被释放出来并被转运至胞浆，最后在宿主细胞内表达外源基因。在鼠伤寒沙门氏菌，有一种由phoP基因（转录激活因子）和PhoQ（传感器激酶）构成的调节子蛋白质。这个操纵子可调控重要毒力功能，包括抵御肽御素cryptdin家族的内源性抗微生物肽。phoP/PhoQ缺失突变体能显著降低对BALB/c小鼠的毒力作用，并作为有效的疫苗应用于很多动物。所以phoP/phoQ缺失突变体的减毒沙门氏菌的菌株，并宿主提供安全保障。目前，phoP/phoQ突变体被广泛地应用于各种药理实验和免疫模型中。旋毛虫有一个复杂的生命周期，涉及肠内和肠外两个阶段，能够诱发粘膜免疫和全身免疫反应，关于旋毛虫入侵的机制还有很多不明确的地方。本实验中的Ts-cystatin编码基因是本实验室在前期工作中，利用感染旋毛虫的猪血清，对旋毛虫生命周期中各个阶段的cDNA文库进行免疫筛选获得的，它是一种高峰度强反应原性基因。本实验旨在通过减毒沙门氏菌真核表达研究该基因对宿主相关免疫应答的影响。虽然沙门氏菌可以携带原核和真核两种质粒，来引起免疫应答，而且许多研究小组已经把注意力集中在在大肠杆菌中的基因表达和收集融合蛋白上。虽然这种方法可以获得大量的蛋白质，但所表达的蛋白质在结构和功能方面可能不同于天然活性蛋白。本实验选择真核表达系统作为该基因的表达载体。本实验检测了体液IgG和粘膜IgA来研究抗体应答，检测脾细胞增殖来分析细胞免疫。为研究该基因对Th1或Th2应答的影响，本实验检测了Th1细胞和Th2细胞因子以及特异性细胞转录因子。此外，利用流式细胞仪检测T淋巴细胞和巨噬细胞变化情况。最后，检查寄生虫数量的变化。结果表明，Ts-cystatin重组减毒沙门氏菌能够诱导机体产生特异性的血清IgG抗体和肠道IgA抗体；能够刺激宿主脾细胞增殖并引起T淋巴细胞和巨噬细胞的变化；能够诱发Th1/Th2混合型免疫反应，并能降低STAT6的转录。Ts-cystatin组肠道肠道成虫回收率增加了15.8％，雌虫繁殖力下降了89％。但是与对照组相比较Ts-cystatin组肌幼虫的数目没有变化。我们的研究结果表明，Ts-cystatin编码基因在旋毛虫抵抗宿主快速排虫反应过程中起重要作用，值得进一步研究。更多还原

【Abstract】 Trichinosis is caused by ingesting undercooked meat containing the larvae of thenematode Trichinella. The infection rate in swine remains high in prevalent areas andposes a great threat to human health. Salmonella-carried targeted genes are extremelyeffective at inducing both cellular and humoral immunity. Administering aSalmonella-carried targeted gene orally can mimic the natural process of Trichinellainfection, which targets the intestinal mucosa, where professional antigen-presentingcells (APCs) are abundant in the intestinal Peyer’s patches (PPs). In S. typhimurium,one such regulon is modulated by the PhoP (transcriptional activator) and PhoQ(sensor kinase) proteins, which are essential to S. typhimurium pathogenesis andsurvival within macrophages. These regulators control the transcription of multipleunlinked phoP-activated and phoP-repressed genes. PhoP/PhoQ null mutants aremarkedly attenuated in BALB/c mice and are effective vaccines in these animals. SophoP/phoQ deletion mutants are attenuated strain of Salmonella and provide securityguarantees for host. Currently, phoP/phoQ mutants were widely used in a variety ofpharmacology experimental studies in mice and immune models.Trichinella has a complex life cycle that involves both enteral and parenteralphases, which stimulate both mucosal and systemic immune responses. Althoughmuch remains unknown about the mechanism of Trichinella invasion, recent studieshave successfully focused on some of the immune regulate antigens of this parasite. Inour previous works, the cDNA encoding a cystatin-like protein (Ts-cystatin) wasidentified by immunoscreening intestinal muscle larvae cDNA libraries with serumfrom pigs experimentally infected with20000T. spiralis muscle larvae. Further,immunoscreening cDNA libraries of all Trichinella life stages that it is a highlyimmunogenic/antigenic gene. The main goal of this work was to determine the abilityof this gene to induce an immune response, when expressed by live attenuatedSalmonella. Although Salmonella is reported to be competent at carrying both prokaryotic and eukaryotic plasmids and then provoking an immune response, manyresearch groups have targeted gene expression in E. coli and have collected the fusionprotein. Although this method can provide a large amount of protein, the expressedprotein may differ from the native active protein in terms of structure and function.We chose a eukaryotic expression system based on several comparisons of theimmunogenicities of two Salmonella administration schemes, which indicated that theeukaryotic expression system is superior. Humoral IgG and mucosal IgA weremeasured to examine the antibody response, and a splenocyte proliferation assay wasperformed to evaluate cellular responses. To explore whether the Th1or Th2responsewas induced, Th1-and Th2-specific cellular transcription factors were examined inaddition to the cytokine profile. Furthermore, changes in T lymphocyte andmacrophage populations were detected by flow cytometry. Lastly, parasitologicalexamination was examined. The results showed that Ts-cystatin induced aTh1/Th2-mixed type of immune response and decreased STAT6transcription. Theintestinal adult recovery increased by15.8%in the Ts-cystatin group, the Ts-cystatingroup fecundity rate was decreased by89%. Furthermore, the number of musclelarvae did not change compared with the control group. In conclusion, our resultssuggest that Ts-cystatin plays an important role in Trichinella resistance to rapidexpulsion by the host and is worth further study.更多还原