本文選題：單增李斯特菌 + InlA蛋白　； 參考：《暨南大學(xué)》2012年碩士論文

【摘要】：目的：制備針對單增李斯特菌(L. monocytogenes)膜表面蛋白的高親和力高特異性的抗體是建立其免疫學(xué)檢測方法的基礎(chǔ)。本研究通過兩種方法來獲得L.monocytogenes特異性的膜表面抗體。①克隆表達(dá)單增李斯特菌特異性的膜表面蛋白InternalinA (InlA),分析其免疫原性，經(jīng)免疫家兔獲得多克隆抗體。②分別用甲醛滅活、加熱滅活的L.monocytogenes全菌以及提取的L.monocytogenes膜表面蛋白免疫小鼠，利用英諾克、威爾斯和格氏李斯特菌進(jìn)行排除篩選，從而制備L.monocytogenes特異性的單抗。為建立其免疫磁珠富集快速檢測方法奠定基礎(chǔ)。 方法：利用生物軟件設(shè)計(jì)單增李斯特菌inlA基因的引物,通過PCR擴(kuò)增出inlA基因,并將其克隆至PET28a(+)原核表達(dá)載體,轉(zhuǎn)化大腸桿菌BL21進(jìn)行優(yōu)化表達(dá)。鎳柱純化表達(dá)產(chǎn)物,質(zhì)譜鑒定重組蛋白, ELISA分析其免疫原性。免疫家兔,制備其多克隆抗體。間接ELISA檢測多抗的效價(jià)及交叉性,免疫熒光分析多抗與單增李斯特菌菌體結(jié)合的特異性。同時(shí)，分別用甲醛滅活和加熱滅活的L.monocytogenes全菌以及L.monocytogenes膜表面蛋白免疫Balb/c小鼠，利用英諾克、威爾斯和格氏李斯特菌進(jìn)行排除篩選，制備L.monocytogenes特異性的單抗。采用ELISA分析所制備抗體的交差性，Western blot分析抗體所識(shí)別的蛋白。并用IP-MS的方法鑒定抗體1B10所識(shí)別的蛋白，原核表達(dá)并純化了該蛋白，Western blot進(jìn)一證實(shí)抗體所識(shí)別蛋白。 結(jié)果：成功表達(dá)了InlA蛋白,融合表達(dá)產(chǎn)物分子量約為92kD,質(zhì)譜鑒定其為InlA蛋白;免疫家兔獲得的抗血清效價(jià)為1∶100000,除與金黃色葡萄球菌約20%的交叉外,與副溶血弧菌等其它病源菌均無交叉;免疫熒光證實(shí)該多抗特異性結(jié)合于單增李斯特菌膜表面,和同種屬的威爾斯李斯特菌不結(jié)合。同時(shí)，制備了三株（1B10、1G3、2C10）L.monocytogenes特異性的單抗和兩株（3G8、1C10）李斯特菌屬特異性的單抗。其中1B10、1G3、2C10只與滅活的單增李斯特菌結(jié)合，與同種屬及其它種屬菌體均不反應(yīng)；其與L.monocytogenes主要的血清型（1/2c、1/2a、4b）都能較好的結(jié)合；它們均識(shí)別72KD左右的一個(gè)蛋白。3G8、1C10能夠識(shí)別單增李斯特菌、英諾克和威爾斯李斯特菌，與其它種屬不存在交叉反應(yīng)。其中只有1B10能較好的識(shí)別L.monocytogenes活菌，并用IP-MS的方法鑒定了該72KD蛋白為MurA蛋白。原核表達(dá)并純化了MurA蛋白，Western blot進(jìn)一步證實(shí)該蛋白為1B10、1G3、2C10所識(shí)別的蛋白。 結(jié)論：成功制備了針對單增李斯特菌體表面具有較好特異性的兔多克隆抗體；同時(shí)制備了單增李斯特菌特異性的單抗，其中MAb1B10與滅活的菌體和活的菌體均能較好的反應(yīng)，，該單抗所識(shí)別的蛋白為MurA蛋白。說明MurA蛋白可以作為特異性檢測L. monocytogenes的一個(gè)很好的靶點(diǎn)。該研究為建立L.monocytogenes免疫磁珠富集快速檢測方法奠定了基礎(chǔ)。
[Abstract]:Aim: to prepare high affinity and high specificity antibody against Lmonocytogenes membrane surface protein of Listeria monocytogenes, which is the basis for the establishment of immunological method for detection of Lmonocytogenes monocytogenes. In this study, two methods were used to obtain the L.monocytogenes specific membrane surface antibody (1. 1) clone and express Listeria monocytogenes specific membrane surface protein (InternalinA). The immunogenicity was analyzed. The polyclonal antibody 2 was inactivated by formaldehyde in rabbits. Mice were immunized with heated inactivated L.monocytogenes and extracted L.monocytogenes membrane surface proteins, and screened by Innoch, Wells and Listeria gravis to prepare L.monocytogenes specific monoclonal antibodies. It lays a foundation for the rapid detection of immunomagnetic bead enrichment. Methods: the primers of inlA gene of Listeria monocytogenes were designed by using biological software. The inlA gene was amplified by PCR and cloned into PET28a () prokaryotic expression vector, and transformed into Escherichia coli BL21 for optimal expression. The expressed product was purified by nickel column, the recombinant protein was identified by mass spectrometry, and its immunogenicity was analyzed by ELISA. Rabbits were immunized and their polyclonal antibodies were prepared. Indirect ELISA was used to detect the titer and cross-section of polyclonal antibodies, and immunofluorescence was used to analyze the specificity of the binding of polyclonal antibodies to Listeria monocytogenes (Listeria monocytogenes). At the same time, Balb/c mice were immunized with formaldehyde inactivated and heated inactivated L.monocytogenes and L.monocytogenes membrane surface protein respectively. L.monocytogenes specific monoclonal antibodies were prepared by exclusion screening with Innoch, Wells and Listeria gravis. The protein recognized by the antibody was analyzed by ELISA and Western blot. The protein recognized by antibody 1B10 was identified by IP-MS. The protein was expressed in prokaryotic and purified by Western blot. Results: the InlA protein was successfully expressed, the molecular weight of the fusion product was about 92 KD, which was identified as InlA protein by mass spectrometry, and the titer of the antiserum was 1: 1 000 000, except for 20% cross with Staphylococcus aureus. There was no cross with other pathogenic bacteria such as Vibrio parahaemolyticus, and immunofluorescence showed that the polyclonal antibody was specifically bound to the membrane surface of Listeria monocytogenes, but not to the same genus Listeria Wales. At the same time, three Monocytogenes Monocytogenes specific McAbs and two Monocytogenes specific Monocytogenes McAbs were prepared. Among them, 1B10G3C10 combined with inactivated Listeria monocytogenes, and did not react with the same species and other species. They could bind well with the main serotype of L.monocytogenes. They could recognize a protein about 72KD. 3G81C10 could identify Listeria monocytogenes. Innoch and Listeria Welsh do not cross-react with other species. Among them, only 1B10 could recognize L.monocytogenes live bacteria well, and the 72KD protein was identified as MurA protein by IP-MS method. MurA protein was expressed and purified by Western blot. It was further confirmed that the protein was recognized by 1B10G _ 3G _ 3H _ 2C _ (10). Conclusion: rabbit polyclonal antibodies against Listeria monocytogenes were successfully prepared, and specific monoclonal antibodies against Listeria monocytogenes were prepared, in which MAb1B10 could react well with inactivated and live bacteria. The protein recognized by the McAb is MurA protein. The results indicate that MurA protein can be used as a good target for specific detection of L. monocytogenes. This study laid a foundation for the rapid detection method of L.monocytogenes immunomagnetic bead enrichment.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378

【參考文獻(xiàn)】

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1 殷月蘭;董慧;焦新安;焦紅梅;顧志強(qiáng);;產(chǎn)單核細(xì)胞李斯特菌actA基因在大腸桿菌中的表達(dá)及其單克隆抗體的研制[J];微生物學(xué)報(bào);2006年06期

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