【作者】 熊国华；

【导师】 曹远银； 曹际娟； 姜永强；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2007， 硕士

【摘要】 单核细胞增生性李斯特菌（Listeria monocytogenes）简称单增李斯特菌，是一种人畜共患的致病菌，属李斯特菌属，它可引起人和动物患脑膜炎、脑炎、败血症、心内膜炎、流产、死胎及脓肿等，发病者死亡率可达30％～40％。近年来已有不少国家报道了由于污染单增李斯特菌发生的食物中毒事件。因此，世界各国政府部门对单增李斯特菌引起的食物中毒越来越重视，纷纷制定了一些新的食品安全法规，并把单增李斯特菌纳入法定强检项目。而葡萄球菌肠毒素（Staphylococcal enterotoxins，SEs）也是重要的人畜共患病原物，能引起人的食物中毒和中毒综合症。这类毒素有很多种，分为SEA、SEB、SEC、SED、SEE、SEG、SHE、SEI和SEJ，其中SEC又分为SEC₁、SEC₂和SEC₃。本试验首先根据NCBI检索李斯特菌溶血素O（hly）序列（登录号：[gi：134116121]），设计引物，常规PCR扩增，并将其产物直接与pMD18-T载体连接，构建克隆测序质粒pMD18-T-hly，进行序列分析鉴定。结果表明，PCR合成的hly基因序列与检索到的hly基因序列完全一致。然后用BamHⅠ和XholⅠ分别双酶切pMD18-T-hly载体和表达载体pET-32a（+），并连接获得重组表达质粒pET-32a（+）-hly。经过双酶切和PCR鉴定正确后，转化到E.coli BL21（DE3）感受态细胞中，构建相应的表达工程菌。利用温度诱导，在30℃诱导培养3.5h后，该工程菌的SDS-PAGE分析显示，有一条相对分子质量约为60kDa的表达蛋白区带，与预期的LLO蛋白的大小相符。经TotalLab20图像软件分析，重组LLO蛋白（rLLO）以包涵体形式存在，表达量约占全菌蛋白的65.48％。根据重组蛋白C端带有6×His标签的特征，用螯合镍离子．次氨基三乙酸（Ni-NTA）的亲和层析柱对重组蛋白进行一步纯化。在变性条件下LLO经pH梯度洗脱，目的蛋白出现在pH4.5洗脱液中，其纯度达96.21％，经亲和纯化方法可获得约2mg／ml的rLLO蛋白。将单增李斯特菌和纯化后的rLLO蛋白、葡萄球菌肠毒素免疫新西兰大白兔分别制备抗单增李斯特菌和抗rLLO、SE的多克隆抗体，获得的免疫血清经正辛酸—硫酸铵分步沉淀法和蛋白A亲和层析纯化，其纯度约达98％，定量抗体浓度约为4mg／mL，间接ELISA法检测抗体效价为1：10⁸以上，WB结果表明所制备的抗原具有很好的免疫原性。纯化后的抗单增李斯特菌、抗SE和抗rLLO多抗，利用胶体金免疫层析技术，采用双抗体夹心法研制了胶体金检测试纸条，用于检测单增李斯特菌、SE和LLO，并对该方法进行特异性、敏感性、稳定性等评价。该检测方法能在5～10min内完成样品检测，多种不同的菌、蛋白及近缘毒素检测评价显示该法特异性良好，检测灵敏性高，SE在奶粉、血清、牛奶、火腿肠等环境样品的检测敏感性也相同。其中单增李斯特菌的敏感性达到10⁶CFU／mL，LLO的敏感性能达到150ng／mL，SEA的敏感性能达到10ng／mL，SEB的敏感性能达到1ng／mL，SEC的敏感性能达到10ng／mL；37℃15天加速试验表明所研制的五种试纸条的稳定性良好，保存期在一年以上。本研究建立的胶体金检测方法灵敏、准确、特异性强，可进一步应用到检验检疫部门对进出口食品中单增李斯特菌和金黄色葡萄球菌的检测实际工作中。此方法建立后，可与经典常规方法互补，预计将在检验检疫、食品工业部门及卫生监控部门具有较广的应用前景，有一定的经济和社会效益。同时，可为食品微生物检验国际方法的修订或增补提供科学依据。更多还原

【Abstract】 Listeria monocytogenes, belonging to Listeria genus, is a kind of pathogenic bacteria whichcan cause human and animal diseases such as meningitis, encephalitis, sepicaemia, endocaritis,abortion, abscess and topical purulent damage, and bring the pregnant woman abortion, stillbirthand etc. The patients’ death rate may reach as high as 30～40%. In recent years, the fact that manycountries have already reported Listeria monocytogenes polluted food toxicosis events paid moreand more serious attention to this bacterium, made a lot of new food security codes and enforcedlegal inspection on Listeria monocytogenes in food. The staphylococcal enterotoxins （SEs） arerectories for the pathogenesis of human and animal illnesses. These toxins are responsible forfood poisoning outbreaks and toxigenic syndrome in humen. The enterotoxins are classifiedserologically into SEA, SEB, SEC, SED, SEE, SEG, SHE, SEI and SEJ, and SEC can be furthercategorized into SEC₁, SEC₂, and SEC₃.Based on the sequence of hly gene in NCBI database under Accession No. gi: 134116121,oligonucleotide primers with mutual overlaps were synthesized based on the optimizedsequences. A 1320 bp gene fragment encoding hly was gained by PCR of two steps, confirmedby sequencing, and subcloned into the expression vector pET32a （+） to construct recombinantexpression vector pET32a （+）-hly. The resulting expression vector pET32a （+）-hly wastransformed into E.coli BL21 （DE3） competent cells and induced at 30℃for 3.5 hours. Aspecific expression band with a relative molecular mass 6kDa was detected by SDS-PAGE ininclusion body form and the protein accounted for 65.48% of total cell protein. The expressedprotein was purified to homogeneity with 96.21% purity using Ni-NTA affinity chromatographymethod under denatured condition, with a yield of 2mg/L of induced culture.In order to develop diagnosis antibody and establish colloidal gold-basedimmunochromatographic assay for rapid detection of L. monocytogenes, Staphylococalenterotoxin and Listeriolysin O （LLO）, the L.monocytogenes, SE and rLLO protein were used toimmunize rabbit for producing the polyclonal antibody against L. monocytogenes, SE or rLLOand then were purified to 98% by caprylic acid and ammonium sulfate precipitation and proteinA affinity chromatography method. The ELISA showed the titter was up to 1:10⁸. The WBshowed the antigen’s immunity was good. Then we have generated rabbit antibodies usingL. monocytogenes or rLLO protein as immunogens. The specificity and titer of the antibodieswere determined by ELISA using L.monocytogenes、SE or rLLO directly coated to the sameantigens. Then anti- L. monocytogenes, anti-SE or anti-LLO antibody was immobilized to a defineddetection zone （as captured antibody） on a porous nitrocellulose membrane, while the anti- L.monocytogenes, anti-SE or anti-LLO antibody was conjugated to colloidal gold particles whichserved as a detection reagent. The L. monocytogenes, SE or rLLO containing sample was addedto the membrane and allowed to react with antibody coated particles. The mixture was thenpassed along the porous membrane by capillary action reacted past the antibody in the detectionzone, which allow binding particles that had antibodies of L. monocytogenes、SE or rLLO boundto their surfaces, giving a red color within this detection zone with an intensity in proportion to L.monocytogenes, SE or LLO concentration. In the absence of L. monocytogenes、SE or LLO, noimmunogold parties were bound to the solid phase antibody. With visual observation, the lowestdetection limit was found to be 10⁶CFU/ml of L. monocytogenes, 150ng/mL of LLO, 10ng/mL ofSEA, 1ng/mL of SEB and 10ng/mL of SEC in less than 10 minutes respectively. These colloidalgold strips no cross-reaction with other bacteria and toxins. The accelerate trail with 37℃in 15days showed the stability of these strips were good and the conservation time would last oneyear.The colloidal gold detection method of L. monocytogenes and SE established in thisexperiment has high sensitivity and specificity. It is quick and accurate. The detection systemestablished in this experiment can be further used in the department of detection and quarantineto do L. monocytogenes and Staphylococcus aureus detection work in the imports and exportsfood. After the method is established, the classical and common method can be completed, andinspection of L. monocytogenes and Staphylococcus aureus can be taken at the molecular biologylevel. This method will have a wide use in the department of inspection and quarantine, foodindustry and sanitation supervision. It will bring a lot of benefits to economy and society.Meanwhile, it may lay the scientific foundation for the revision or supplement of theinternational detection method of food microbiology.更多还原