乳源金葡菌主要肠毒素基因的检测、表达及其抗体制备

发布时间：2018-05-27 00:08

本文选题：乳源金葡菌 + 主要肠毒素基因型　；参考：《安徽农业大学》2012年硕士论文

【摘要】：金葡菌肠毒素(Staphylococcal Enterotoxin, SE)是引起人类食物中毒和葡萄球菌胃肠炎的主要原因。SE共有18个基因类型，不同SE基因的检出率与菌株分离地域和宿主来源有关。针对合肥地区乳源金葡菌肠毒素基因型分布状况尚不清楚的现状，本研究在采用PCR方法检测56株合肥分离株10种SE基因，确定其主要基因型的基础上，利用大肠杆菌原核表达系统成功表达了主要肠毒素SEA和SEG蛋白，并制备了其特异性抗体。研究结果为进一步建立金葡菌肠毒素的免疫学检（监）测方法奠定了基础，对保障乳制品的安全，防止SE食物中毒具有一定指导意义。为了明确合肥地区乳源金葡菌肠毒素基因型的分布状况，根据GenBank中收录的各型SE基因序列(SEA、SEB、SEC、SED、SEE、SEG、SEH、SEI、SEM和SEN)，设计10对特异性引物，，对56株乳源金葡菌合肥分离株进行10种SE基因PCR检测。结果显示56株测试菌中有53株携带SE基因，总携带率为94.64%，各型肠毒素基因的携带率分别为SEA41.07%、SEB16.07%、SEC19.64%、SED3.57%、SEG91.07%、SEH5.36%、SEI17.86%、SEM5.36%和SEN24.43%，未检测到SEE基因。SE阳性菌株可同时携带两种或两种以上的SE基因，共组成49种肠毒素基因型，但以SEA-SEG为其主要肠毒素基因型。结果提示，应建立一种敏感、快速和特异的免疫学方法，加强对合肥地区原料乳中SEA和SEG的检（监）测。为了获得SEA和SEG蛋白检测原和免疫原，根据金葡菌ATCC25923的SEA和SEG基因序列，设计2对特异性引物，PCR扩增出全长SEA基因(774bp)和切除信号肽的SEG基因(702bp)。分别将PCR扩增产物克隆至pAML-c4X质粒，构建出原核表达重组质粒pAML-c4X-SEA和pAML-c4X-SEG。诱导表达的可溶性重组蛋白经亲和层析柱纯化后，得到纯度分别为98.32%和97.69%、浓度分别为1.0mg/mL和1.5mg/mL的重组SEA和SEG蛋白，它们均满足作为检测原和免疫原的要求。为了制备SEA和SEG蛋白检测抗体，将纯化的重组SEA和SEG蛋白分别与双相乳化佐剂充分乳化制成免疫原，分别免疫家兔，二次免疫后第14天抗SEA抗体的ELISA效价和琼扩效价分别高达1:6553600和1:64，抗SEG抗体的ELISA效价和琼扩效价分别高达1:3276800和1:32，它们纯化后均可作为检测抗体使用。
[Abstract]:Staphylococcus aureus enterotoxin (Staphylococcal Enterotoxin, SE) is the main cause of human food poisoning and staphylococcal gastroenteritis,.SE has 18 genetic types. The detection rate of different SE genes is related to the region of isolation and the host source. The distribution of staphylococcal enterotoxin genotypes in Ruyuan, Hefei, is not yet clear. In this study, on the basis of the determination of the main genotypes of 10 SE genes in 56 Hefei isolates, the main enterotoxin SEA and SEG proteins were successfully expressed by the Escherichia coli prokaryotic expression system, and their specific antibodies were prepared. The results of the study lay a foundation for the further establishment of the immunoassay method for the establishment of Staphylococcus aureus enterotoxin. It lays a foundation for ensuring the safety of dairy products and preventing SE food poisoning.
In order to identify the distribution of staphylococcal enterotoxin genotypes in Ruyuan, Hefei, 10 pairs of specific primers were designed according to the sequence of SE gene sequences included in GenBank (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEM and SEN), and 10 kinds of 56 strains of Staphylococcus aureus Hefei isolates were detected. The results showed that 53 of the 56 tested strains were carried. The total carrying rate of SE gene is 94.64%, and the carrying rates of all types of enterotoxin genes are SEA41.07%, SEB16.07%, SEC19.64%, SED3.57%, SEG91.07%, SEH5.36%, SEI17.86%, SEM5.36% and SEN24.43%. No SEE gene.SE positive strains can carry two or more than two kinds of genes simultaneously, and 49 types of enterotoxin genotypes are formed. The main enterotoxin genotypes. The results suggest that a sensitive, rapid and specific immunological method should be established to strengthen the detection of SEA and SEG in raw milk in Hefei.
In order to obtain SEA and SEG protein to detect the original and immunogen, 2 pairs of specific primers were designed on the basis of the SEA and SEG gene sequences of Staphylococcus aureus ATCC25923. The SEG gene (774bp) and the SEG gene (702bp) were amplified by PCR, and the PCR amplified products were cloned to the pAML-c4X particles. The soluble recombinant protein induced by X-SEG. was purified by affinity chromatography column, and the recombinant SEA and SEG protein with the purity of 98.32% and 97.69% respectively, and the concentration of 1.0mg/mL and 1.5mg/mL respectively, were all satisfied as the requirements for the detection of the original and immunogen.
In order to prepare SEA and SEG protein to detect antibodies, the purified recombinant SEA and SEG protein were fully emulsified with the biphasic emulsification adjuvant and immunogen respectively. The ELISA titer and agar expansion potency of anti SEA antibody were up to 1:6553600 and 1:64 respectively after two times of immunization. The ELISA titer and agar expansion titer of anti SEG antibody were up to 1:3276, respectively. At 800 and 1:32, they could be used as antibodies for detection after purification.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.1

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