本文關(guān)鍵詞： EHEC O157 Stx1B Stx2B eae 原核表達(dá) 單克隆抗體　出處：《東北農(nóng)業(yè)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 腸出血性大腸桿菌(EHEC)是一種重要的食源性病原菌,自1975年被首次分離接著被確認(rèn)為致病菌以來,世界各地包括中國在內(nèi)都有不同規(guī)模的暴發(fā)流行,可引起出血性或非出血性腹瀉、出血性結(jié)腸炎(HC)、溶血性尿毒綜合征(HUS)、血栓性血小板減少性紫癜(TTP)等全身性并發(fā)癥。O157是EHEC的代表菌株,其主要毒力特征之一是引起腸上皮細(xì)胞出現(xiàn)黏附與脫落(attaching and effacing, AE)損傷,編碼AE損傷的基因都位于細(xì)菌染色體上LEE致病島內(nèi),其中最重要的就是eae基因,編碼一個(gè)被稱為緊密素的外膜蛋白。EHEC O157另外一個(gè)毒力因子是由插入到染色體上的原噬菌體編碼的志賀毒素(Stx),它能使感染機(jī)體器官發(fā)生病理變化,或者繼發(fā)其他的臨床癥狀甚至引起死亡。大量研究證明,緊密素和志賀毒素Stx是可以用于開發(fā)疫苗以預(yù)防EHEC感染的重要免疫原。 本試驗(yàn)利用PCR技術(shù)從大腸桿菌EDL933全基因組中克隆了Stx1B、Stx2B、eae三段基因,經(jīng)序列分析,結(jié)果與參考序列同源性達(dá)99%以上。將產(chǎn)物克隆到原核表達(dá)載體pGEX-6P-1,構(gòu)建重組表達(dá)質(zhì)粒pGEX-Stx1B、pGEX-Stx2B、pGEX-eae,轉(zhuǎn)入大腸桿菌BL21中,經(jīng)IPTG誘導(dǎo),實(shí)現(xiàn)了重組融合蛋白的高效表達(dá),表達(dá)量分別約占菌體總蛋白的27%、27.7%和19.5%,且均主要以包涵體形式表達(dá),通過切膠的方法純化目的蛋白。 純化蛋白免疫BABL/c小鼠,利用細(xì)胞融合技術(shù),經(jīng)間接ELISA篩選和3次細(xì)胞亞克隆,Stx1B、Stx2B、eae分別獲得5、5、2株雜交瘤細(xì)胞,分別命名為1G11、2H8、1B10、3A6、4B1;1B3、4B4、2C3、3D1、1C2;2D4、3B4。經(jīng)單克隆抗體亞類試劑盒鑒定,2D4為IgM,其他均為IgG類抗體。12株雜交瘤細(xì)胞染色體數(shù)為103±5,明顯多于SP2/0骨髓瘤細(xì)胞65～75條。Western blotting表明1G11、2H8、1B10,1B3、4B4、2C3,2D4、3B4與相應(yīng)的GST-Stx1B、GST-Stx2B、GST-eae融合蛋白反應(yīng)均能產(chǎn)生特異性條帶,而與空載體誘導(dǎo)后的GST標(biāo)簽蛋白不反應(yīng)。制備了1G11、2H8腹水單抗,采用辛酸-飽和硫酸銨法純化后,經(jīng)SDS-PAGE電泳分析IgG重鏈和輕鏈區(qū)帶明顯,幾乎沒有雜帶。ELISA疊加試驗(yàn)可知,1G11、2H8、1B10抗同一抗原表位,1B3、4B4、2C3抗同一抗原表位,2D4、3B4抗不同的抗原表位。12株雜交瘤細(xì)胞在體外連續(xù)傳代3個(gè)月和凍存后再復(fù)蘇均不影響抗體分泌的穩(wěn)定性;與沙門菌、大腸桿菌、牛結(jié)核桿菌、李氏桿菌、鏈球菌、金黃色葡萄球菌均無交叉反應(yīng)。親和力分析表明,1G11、2H8、1B10,1B3、4B4、2C3,2D4、3B4 8株單抗的親和常數(shù)均在107～109M-1之間,親和力較高,為這些單抗敏感、穩(wěn)定和快捷的檢測應(yīng)用提供了必要的保證�？傊�,12株單抗的成功制備為EHEC O157病原的快速、特異性診斷提供了診斷試劑,同時(shí)也為該病致病機(jī)理和特異性治療的進(jìn)一步研究提供物質(zhì)基礎(chǔ),為成功研制O157疫苗積累了數(shù)據(jù)。
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen, which has been isolated for the first time since 1975 and then confirmed as a pathogenic bacteria. All over the world, including China, there are outbreaks of different scales, which can cause hemorrhagic or non-hemorrhagic diarrhea, hemorrhagic colitis, hemorrhagic colitis, hemolytic uremic syndrome (HUSS). Thrombotic thrombocytopenic purpura (TTP) and other systemic complications. O157 is the representative strain of EHEC. One of its main virulence characteristics is to cause adhesion and abscission of and effusion (AEs) injury in intestinal epithelial cells. The genes encoding AE damage are located in the LEE pathogenicity island on the bacterial chromosome, the most important of which is the eae gene. Another virulence factor that encodes an outer membrane protein, EHEC O157, called compaction, is the Shiga toxin Stx encoded by the prophage inserted into the chromosome. It can cause pathological changes in infected organs, or other clinical symptoms or even death. A large number of studies prove that. Titin and Shigella toxin Stx are important immunogens that can be used to develop vaccines to prevent EHEC infection. In this study, three segments of Stx1BX Stx2BUE eae gene were cloned from the whole genome of Escherichia coli EDL933 by PCR technique and sequenced. Results the homology with the reference sequence was over 99%. The product was cloned into the prokaryotic expression vector pGEX-6P-1 and the recombinant expression plasmid pGEX-Stx1BnpGEX-Stx2B was constructed. PGEX-eaewas transferred into Escherichia coli BL21 and induced by IPTG, the recombinant fusion protein was highly expressed, which accounted for about 27% of the total bacterial protein. Both 27.7% and 19.5g were expressed in the form of inclusion body, and the target protein was purified by gumming method. The purified protein was used to immunize BABL/c mice. By indirect ELISA screening and three cell subclones Stx2Beae, 5 BABL/c mice were obtained. Two hybridoma cells were named as 1G11O2H8B103A6A6B1; 1B _ 3N _ 4B _ 4N _ 2C _ 3C _ 3N _ 3D _ 1C _ 2; The chromosome number of the hybridoma cells was 103 鹵5. The chromosome number of the other hybridoma cells was 103 鹵5, which was identified as IgM by monoclonal antibody subclass kit. There were more than 6575 SP2/0 myeloma cells. Western blotting showed that 1G112H8, 1B10, 1B3, 4B4, 2C3D4. 3B4 reacted with the corresponding GST-Stx1BU GST-Stx2BU GST-eae fusion protein to produce specific bands. The 1G112H8 ascites monoclonal antibody was prepared and purified by octanoic acid-saturated ammonium sulfate method. The IgG heavy chain and light chain bands were obviously analyzed by SDS-PAGE electrophoresis. There was almost no heteroband. Elisa superposition test showed that 1G112H8H8 1B10 could resist the same antigen epitope 1B3. The antigenic epitopes of 4B4O2C3 against different epitopes. 12 hybridoma cells were subcultured for 3 months in vitro and resuscitated after cryopreservation did not affect the stability of antibody secretion. There was no cross reaction with Salmonella, Escherichia coli, Mycobacterium bovis, Listeria, Streptococcus, Staphylococcus aureus. The affinity constants of the McAbs of 2C3D4D4B48 were between 107 and 109M-1, and they were sensitive to these McAbs because of their high affinity. The stable and rapid detection and application provided the necessary guarantee. In a word, the successful preparation of 12 McAbs provided a diagnostic reagent for the rapid and specific diagnosis of EHEC O157. It also provides material basis for further research on the pathogenesis and specific treatment of the disease, and accumulates data for the successful development of O157 vaccine.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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