幽门螺杆菌UreB与大肠杆菌LTB融合蛋白的表达及生物活性研究

目的 通过基因工程方法构建并表达幽门螺杆菌尿素酶B亚单位(UreB)和大肠杆菌不耐热肠毒素B亚单位(LTB)以基因形式的融合蛋白,并对其生物活性进行初步研究,为幽门螺杆菌疫苗的研究奠定基础。方法 用PCR方法从本室构建的融合基因克隆载体扩增出融合基因2 040bp的片段,将融合基因插入原核表达载体pET-11-c中。结果 经全自动测序仪测序,SDS-PAGE和免疫印迹以及N端氨基酸测序分析,证实融合蛋白已在BL21中表达,初步纯化后通过动物实验和酶联免疫吸附试验证实其相应的免疫原性和免疫反应性,以及其中LTB成分和GM1结合的特性。结论 融合蛋白表达方式为幽门螺杆菌分子内佐剂疫苗的研究奠定了基础。     

幽门螺杆菌根除治疗对肝硬化患者血氨水平的影响

目的虽然肝性脑病的发病机制迄今尚未完全阐明,但氨中毒学说始终在其发生中占有重要地位。幽门螺杆菌(Helicobacter pylori,H.pylori)具有强大的尿素酶活性,在胃内可分解尿素产氨,因此可能影响到肝硬化患者的血氨水平。本研究探讨了H.pylori根除治疗对肝硬化患者血氨水平的影响。方法对34例根据病史、临床表现、超声及实验室检查确诊为肝硬化的住院患者,行抗H.pylori抗体血清学检测、快速尿素酶试验或尿素呼气试验。对H.pylori阳性患者采用三联疗法(奥美拉唑20mg,阿莫西林1.0g,克拉霉素0.5g,均每日2次,疗程1周。)根除H.pylori治疗,并比较治疗前后血氨浓度。结果肝硬化患者中H.pylori的感染率为58.82%。20例H.pylori感染患者基础血氨水平明显高于14例未感染H.pylori患者(1887±332μg/L对1346±124μg/L,P<0.01),H.pylori阳性的肝硬化患者于根除治疗后血氨水平明显下降(1887±332μg/L对1590±195μg/L,P<0.01)。结论H.pylori感染可影响肝硬化患者的血氨水平,并在肝性脑病的发生中起一定作用。     

Indene Compounds Synthetically Derived from Vitamin D Have Selective Antibacterial Action on Helicobacter pylori

Helicobacter pylori infects the human stomach and is closely linked with the development of gastric cancer. When detected, this pathogen can be eradicated from the human stomach using wide‐spectrum antibiotics. However, year by year, H. pylori strains resistant to the antibacterial action of antibiotics have been increasing. The development of new antibacterial substances effective against drug‐resistant H. pylori is urgently required. Our group has recently identified extremely selective bactericidal effects against H. pylori in (1R,3aR,7aR)‐1‐[(1R)‐1,5‐dimethylhexyl]octahydro‐7a‐methyl‐4H‐inden‐4‐one (VDP1) (otherwise known as Grundmann's ketone), an indene compound derived from the decomposition of vitamin D₃ and proposed the antibacterial mechanism whereby VDP1 induces the bacteriolysis by interacting at least with PtdEtn (dimyristoyl‐phosphatidylethanolamine [di‐14:0 PtdEtn]) retaining two 14:0 fatty acids of the membrane lipid constituents. In this study, we synthesized new indene compounds ((1R,3aR,7aR)‐1‐((2R,E)‐5,6‐dimethylhept‐3‐en‐2‐yl)‐7a‐methyloctahydro‐4H‐inden‐4‐one [VD2‐1], (1R,3aR,7aR)‐1‐((S)‐1‐hydroxypropan‐2‐yl)‐7a‐methyloctahydro‐1H‐inden‐4‐ol [VD2‐2], and (1R,3aR,7aR)‐7a‐methyl‐1‐((R)‐6‐methylheptan‐2‐yl)octahydro‐1H‐inden‐4‐ol [VD3‐1]) using either vitamin D₂ or vitamin D₃ as materials. VD2‐1 and VD3‐1 selectively disrupted the di‐14:0 PtdEtn vesicles without destructing the vesicles of PtdEtn (dipalmitoyl‐phosphatidylethanolamine) retaining two 16:0 fatty acids. In contrast, VD2‐2, an indene compound lacking an alkyl group, had no influence on the structural stability of both PtdEtn vesicles. In addition, VD2‐1 and VD3‐1 exerted extremely selective bactericidal action against H. pylori without affecting the viability of commonplace bacteria. Meanwhile, VD2‐2 almost forfeited the bactericidal effects on H. pylori. These results suggest that the alkyl group of the indene compounds has a crucial conformation to interact with di‐14:0 PtdEtn of H. pylori membrane lipid constituents whereby the bacteriolysis is ultimately induced.     

HpaA蛋白在幽门螺杆菌感染诊断中的应用

目的建立检测血清HpaA抗体的ELISA间接法,探讨以重组蛋白作为抗原在诊断H.pylori感染中的价值。方法将从临床分离菌株中获得的HpaA基因在大肠杆菌中表达,用Ni2+-NTA柱纯化表达的HpaA作为抗原,建立检测血清HpaA抗体的ELISA间接法,与诊断标准比较评价其应用的可行性。结果经超声破碎后,用SDS-PAGE分析显示,HpaA蛋白主要存在于上清中,纯化抗原检测临床标本中HpaA抗体的敏感性和特异性分别为100.0%和90.1%。结论以重组蛋白为抗原初步建立检测血清HpaA抗体的ELISA间接法敏感性、特异性高,为制备商品化的试剂盒奠定了基础。     

三氮唑类衍生物的合成及其对幽门螺杆菌体外抑制活性研究

设计并合成了15个未曾见文献报道的1-(2-(4-取代)苯氧乙基)-1H-1,2,3-三氮唑衍生物,并通过NMR、IR、MS确认了它们的结构.幽门螺旋杆菌(HP)体外抑制活性测试结果表明,4个化合物对16株HP表现出一定的抑制活性.     

重组幽门螺杆菌粘附素的纯化工艺

目的纯化大肠杆菌表达的重组幽门螺杆菌粘附素。方法将表达HpaA蛋白的工程菌经高压均质机破菌获得包涵体,经洗涤、变性、复性,Q Sepharose High Performance阴离子交换层析和亲和层析分离纯化。采用SDS-PAGE和HPLC检测纯度,用Western blot检测其抗原性。结果纯化后HpaA蛋白纯度高达95％以上,具有良好的抗原性。结论建立了从包涵体中获得高纯度HpaA纯化工艺,为进一步的研究打下了基础。