【作者】 田国明；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 预防兽医学， 2007， 硕士

【摘要】 单核细胞增多性李斯特菌(Listeria monocytogenes,以下简称Lm或单增李斯特菌)为短小的革兰氏阳性无芽胞兼性厌氧杆菌,是食源性人畜共患病原菌。孕妇、小孩、老年人及免疫力低下人群易感,常表现为脑膜脑炎、脑炎、脓血症、流产等。在主要食源性致病菌中,单增李斯特菌引起的死亡率最高,20世纪90年代以来,WHO将其列为四大食源性疾病致病菌之一。该菌在食品加工和生产过程中可以通过多种途径污染食品,对消费者健康构成潜在威胁。因此,建立快速、敏感、特异、简单的方法用于食品及其加工环境中该菌的检测,对控制和提高食品的卫生质量具有重要意义。本研究选择单增李斯特菌表面蛋白LMO1847为研究对象,设计其编码基因的特异性引物,PCR扩增目的片段后插入原核表达载体pET30a,获得的重组质粒命名为pLMO1847,重组质粒转化大肠杆菌BL21感受态细胞,诱导表达并分析其产物。结果表明,LMO1847基因片段获得了融合表达,可溶性分析发现该蛋白以可溶性和包涵体两种形式存在。大量表达并纯化该蛋白后,免疫家兔制备多克隆抗体,该多抗的效价高达1：25600,表明该蛋白具有良好的免疫原性。以纯化的LMO1847蛋白作为包被抗原,鼠源抗单增李斯特菌全菌多抗作为一抗,ELISA结果显示纯化蛋白与全菌多抗具有良好的反应性。以单增李斯特菌全菌作为包被抗原,LMO1847的多抗作为一抗,也呈现良好的反应活性。Western-blotting检测显示,纯化蛋白制备的多抗能够与单增李斯特菌外膜蛋白提取物中的LMO1847发生特异性反应。DOT-ELISA结果表明该多抗可与李斯特菌属的不同种发生交叉反应,但不与其它属细菌发生交叉反应,提示该蛋白可能为李斯特菌属特异性蛋白。单增李斯特菌在高盐、酸性、以及高温或低温的短期应激后(3小时),除5.5-NaCl处理组在1小时后LMO1847表达量略有下降外,42℃、pH5.5、pH3.0处理组表达基本稳定。但酸处理后,DOT-ELISA检测不到菌体表面的LMO1847蛋白。高盐(NaCl 5.5%-8%)条件下生长时,虽然细菌外膜蛋白LMO1847表达量有所下降,但仍可通过DOT-ELISA检测到。低温(4-15℃)条件下生长时,该蛋白表达量明显降低,DOT-ELISA显示4℃时可能由于表达量太低而无法在菌体表面检测到该蛋白。该蛋白可作为高盐腌制及高温消毒食品中李斯特菌的检测靶抗原,但不适宜检测低温保存或酸性食品中李斯特菌的污染。微生物快速检测的另一领域为针对特异性基因的分子检测。本试验基于近几年发展的PNA(肽核酸)技术,结合FISH(荧光原位杂交)技术,初步建立了食品中常见致病性微生物,单增李斯特菌的快速检测方法。首先通过生物信息学的方法,从NCBI数据库下载李斯特菌属23S rDNA序列,运用DNASTAR软件进行比较分析,结合PNA探针设计的原则,设计了两条针对李斯特菌23SrRNA的N末端标记荧光素的PNA探针,利用其中一条PNA探针建立了液相和固相两种模式的荧光原位杂交检测方法,并优化了两种模式下的杂交条件。液相杂交最佳条件为,50：50酒精／PBS固定,浓度为107CFU/ml的菌体于(20mM Tris pH9.0,100 mM NaCl,0.5%SDS,300pmol/mlPNA)的杂交缓冲液中55。C杂交1.5小时,洗涤后涂片观察。固相杂交的最佳条件为,浓度为106-107CFU/ml的菌体涂片,火焰固定后于80%酒精中浸泡15min,风干,用含500pmol/ml PNA (0.2%TritonX-100)的杂交缓冲液55℃杂交1.5小时。分别用两种检测模式对李斯特菌属及部分其他属细菌进行检测,其中2株单增李斯特菌参考株、10株分离株,6个不同种的非单增李斯特菌各1株,其他属细菌4株,共22株。两种模式检测结果一致,与分离鉴定结果吻合,但不同分离株杂交效率存在差异。该探针可与李斯特菌属的5个种杂交,不与其它属细菌杂交,与理论预测符合,对李斯特菌属细菌的检测具有一定的应用价值。本研究为开发基于特异、高亲和力的抗体包被磁珠,通过免疫磁珠快速分离、富集食品中单增李斯特菌,结合PNA荧光原位杂交技术或其它方法,为食品中单增李斯特菌快速检测系统的构建奠定了基础。更多还原

【Abstract】 Listeria monocytogenes is a Gram-positive, rod-shaped, facultative anaerobic, non-spore-forming bacterium. As a facultative intracellular pathogen it can cause listeriosis, a severe food-borne infection in humans and animals leading to sydromes such as meningitis, encephalitis, and sepsis in immunocompromised individuals, neonates, the eldly, or spontaneous abortion in pregnant women. Listeia monocytogenes was reported to cause the highest mortality among the food-borne pathogens. World health organization considered it as one of the four most severe food-borne pathogens from 1990S. Contamination of foodstuff by L. monosytogenes can be due to the processing environments. So it is important to set up a rapid, sensitive, differential, and simple detection method to supervise and control its contamination in foods and processing environments.In this study, we selected the surface protein LMO1847 of Listeria monocytogenes as the target based on the research of cell wall subproteome of listeria monocytogenes. The whole gene fragment LMO1847 was amplified from Listeia monocytogenes genome and was inserted between EcoR I and Xho I sites of prokaryotic expression vector pET30a to construct the recombinant plasmid pLMO1847 which was then transferred into E.coli BL21. Expression of the recombinant protein was induced by IPTG and analysed by SDS-PAGE. Polyclonal antibody was prepared by immunizing the rabbits with the purified protein and the antibody titer was as high as 1:25600.Prokaryotic expression product LMO1847 reacted with the antibody of the whole Listeria monocytogenes from mice. The polyclonal antibody of LMO1847 could also react with the whole cells of Listeria monocytogenes. The cellullar LMO1847 extracts from Listeria monocytogenes could be recognized by rabbit polyclonal antibody of LMO1847 by western blotting without any non-special bands. The cross reaction of polyclonal antibody of LMO1847 was analysed by DOT-ELISA, and the polyclonal antibody could react with all Listeria species but not with any unrelated bacterium. The protein LMO1847 might be the specific protens in the Listeria genus. Listeria monocytogenes was held in stressful environments such as salt, temperature, and acid for a short duration (3 hours), the expression of LMO1847 were investigated by reaction with its antibody by ELISA or blotting. Expression of LMO1847 was not affected under stress conditions such as 42℃, pH5.5 and pH3.0. Reduced expression was seen upon its exposure to 5.5%NaCl for an hour. During short period exposure to acid stress environment, LMO1847 in the surface of whole cells was not detectable. Expression of LMO1847 was reduced and be detectable by DOT-ELISA when L. monocytogenes grown in high salt concentration (5.5%-8% NaCl). The expresson was also reduced when grown at low temperature (4-15℃). We suppose that the antibody of LMO1847 could detect Listeria from osmotically and high temperature stressed environments, but might not be suitable to detect cells from cold and acid stressed envioments.The other area for rapid detection of pathogens is moleculer detection targeting special genes. In this study, a new rapid detection method for Listeria monocytogenes was approached based on the technology of peptide nucleic acid (PNA) and fluorescence in situ hybridization (FISH). Two peptide nucleic acid probes labelled with fluorescein at N terminal were designed according to the probe designing principle and sequence analysis of 23S rRNA of Listeria spp. Two hybridization forms, liquid-phase PNA FISH and slide-based PNA FISH, were set up using one PNA probe. The probe reactivity was evaluated against 16 Listeria strains and 4 closely related strains base on the two hybridization forms after optimization of the hybridization conditions. The two hybridization forms were accordant with each other and also agreed with theoretical prediction. This probe could be used to detect 5 Listeia species and had no cross reaction to any other strains tested.Our study provides some foundations for enrichment of listerial cells using specific antibodies for quick identification of L. moncytogenes by PNA-FISH or other methods.更多还原