单增李斯特菌特异性的膜表面蛋白的抗体的制备

发布时间：2018-05-15 02:00

本文选题：单增李斯特菌 + InlA蛋白　；参考：《暨南大学》2012年硕士论文

【摘要】：目的：制备针对单增李斯特菌(L. monocytogenes)膜表面蛋白的高亲和力高特异性的抗体是建立其免疫学检测方法的基础。本研究通过两种方法来获得L.monocytogenes特异性的膜表面抗体。①克隆表达单增李斯特菌特异性的膜表面蛋白InternalinA (InlA),分析其免疫原性，经免疫家兔获得多克隆抗体。②分别用甲醛灭活、加热灭活的L.monocytogenes全菌以及提取的L.monocytogenes膜表面蛋白免疫小鼠，利用英诺克、威尔斯和格氏李斯特菌进行排除筛选，从而制备L.monocytogenes特异性的单抗。为建立其免疫磁珠富集快速检测方法奠定基础。方法：利用生物软件设计单增李斯特菌inlA基因的引物,通过PCR扩增出inlA基因,并将其克隆至PET28a(+)原核表达载体,转化大肠杆菌BL21进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白, ELISA分析其免疫原性。免疫家兔,制备其多克隆抗体。间接ELISA检测多抗的效价及交叉性,免疫荧光分析多抗与单增李斯特菌菌体结合的特异性。同时，分别用甲醛灭活和加热灭活的L.monocytogenes全菌以及L.monocytogenes膜表面蛋白免疫Balb/c小鼠，利用英诺克、威尔斯和格氏李斯特菌进行排除筛选，制备L.monocytogenes特异性的单抗。采用ELISA分析所制备抗体的交差性，Western blot分析抗体所识别的蛋白。并用IP-MS的方法鉴定抗体1B10所识别的蛋白，原核表达并纯化了该蛋白，Western blot进一证实抗体所识别蛋白。结果：成功表达了InlA蛋白,融合表达产物分子量约为92kD,质谱鉴定其为InlA蛋白;免疫家兔获得的抗血清效价为1∶100000,除与金黄色葡萄球菌约20%的交叉外,与副溶血弧菌等其它病源菌均无交叉;免疫荧光证实该多抗特异性结合于单增李斯特菌膜表面,和同种属的威尔斯李斯特菌不结合。同时，制备了三株（1B10、1G3、2C10）L.monocytogenes特异性的单抗和两株（3G8、1C10）李斯特菌属特异性的单抗。其中1B10、1G3、2C10只与灭活的单增李斯特菌结合，与同种属及其它种属菌体均不反应；其与L.monocytogenes主要的血清型（1/2c、1/2a、4b）都能较好的结合；它们均识别72KD左右的一个蛋白。3G8、1C10能够识别单增李斯特菌、英诺克和威尔斯李斯特菌，与其它种属不存在交叉反应。其中只有1B10能较好的识别L.monocytogenes活菌，并用IP-MS的方法鉴定了该72KD蛋白为MurA蛋白。原核表达并纯化了MurA蛋白，Western blot进一步证实该蛋白为1B10、1G3、2C10所识别的蛋白。结论：成功制备了针对单增李斯特菌体表面具有较好特异性的兔多克隆抗体；同时制备了单增李斯特菌特异性的单抗，其中MAb1B10与灭活的菌体和活的菌体均能较好的反应，，该单抗所识别的蛋白为MurA蛋白。说明MurA蛋白可以作为特异性检测L. monocytogenes的一个很好的靶点。该研究为建立L.monocytogenes免疫磁珠富集快速检测方法奠定了基础。
[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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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