本文選題：乳源金葡菌 + 主要腸毒素基因型��； 參考：《安徽農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：金葡菌腸毒素(Staphylococcal Enterotoxin, SE)是引起人類食物中毒和葡萄球菌胃腸炎的主要原因。SE共有18個基因類型，不同SE基因的檢出率與菌株分離地域和宿主來源有關(guān)。針對合肥地區(qū)乳源金葡菌腸毒素基因型分布狀況尚不清楚的現(xiàn)狀，本研究在采用PCR方法檢測56株合肥分離株10種SE基因，確定其主要基因型的基礎(chǔ)上，利用大腸桿菌原核表達(dá)系統(tǒng)成功表達(dá)了主要腸毒素SEA和SEG蛋白，并制備了其特異性抗體。研究結(jié)果為進(jìn)一步建立金葡菌腸毒素的免疫學(xué)檢（監(jiān)）測方法奠定了基礎(chǔ)，對保障乳制品的安全，防止SE食物中毒具有一定指導(dǎo)意義。 為了明確合肥地區(qū)乳源金葡菌腸毒素基因型的分布狀況，根據(jù)GenBank中收錄的各型SE基因序列(SEA、SEB、SEC、SED、SEE、SEG、SEH、SEI、SEM和SEN)，設(shè)計10對特異性引物，，對56株乳源金葡菌合肥分離株進(jìn)行10種SE基因PCR檢測。結(jié)果顯示56株測試菌中有53株攜帶SE基因，總攜帶率為94.64%，各型腸毒素基因的攜帶率分別為SEA41.07%、SEB16.07%、SEC19.64%、SED3.57%、SEG91.07%、SEH5.36%、SEI17.86%、SEM5.36%和SEN24.43%，未檢測到SEE基因。SE陽性菌株可同時攜帶兩種或兩種以上的SE基因，共組成49種腸毒素基因型，但以SEA-SEG為其主要腸毒素基因型。結(jié)果提示，應(yīng)建立一種敏感、快速和特異的免疫學(xué)方法，加強對合肥地區(qū)原料乳中SEA和SEG的檢（監(jiān)）測。 為了獲得SEA和SEG蛋白檢測原和免疫原，根據(jù)金葡菌ATCC25923的SEA和SEG基因序列，設(shè)計2對特異性引物，PCR擴(kuò)增出全長SEA基因(774bp)和切除信號肽的SEG基因(702bp)。分別將PCR擴(kuò)增產(chǎn)物克隆至pAML-c4X質(zhì)粒，構(gòu)建出原核表達(dá)重組質(zhì)粒pAML-c4X-SEA和pAML-c4X-SEG。誘導(dǎo)表達(dá)的可溶性重組蛋白經(jīng)親和層析柱純化后，得到純度分別為98.32%和97.69%、濃度分別為1.0mg/mL和1.5mg/mL的重組SEA和SEG蛋白，它們均滿足作為檢測原和免疫原的要求。 為了制備SEA和SEG蛋白檢測抗體，將純化的重組SEA和SEG蛋白分別與雙相乳化佐劑充分乳化制成免疫原，分別免疫家兔，二次免疫后第14天抗SEA抗體的ELISA效價和瓊擴(kuò)效價分別高達(dá)1:6553600和1:64，抗SEG抗體的ELISA效價和瓊擴(kuò)效價分別高達(dá)1:3276800和1:32，它們純化后均可作為檢測抗體使用。
[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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378.1

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