【摘要】：目的：hly是編碼單增李斯特菌特有的溶血素蛋白LLO的基因,克隆hly表達(dá)LLO蛋白并制備單增李斯特菌特異性的單克隆抗體,為單增李斯特菌特異性檢測方法的建立奠定基礎(chǔ)。 方法：以單增李斯特菌的DNA為模板,通過PCR擴增出hly基因,與PET28a(+)原核表達(dá)載體連接,經(jīng)測序鑒定后,轉(zhuǎn)化大腸桿菌BL21,并誘導(dǎo)表達(dá)。經(jīng)表達(dá)條件優(yōu)化,大量誘導(dǎo)表達(dá),用鎳柱純化。通過SDS-PAGE和westernblotting鑒定表達(dá)的蛋白,通過溶血試驗鑒定其溶血活性。用純化的表達(dá)產(chǎn)物免疫Balb/c小鼠,利用雜交瘤技術(shù)制備抗LLO的單克隆抗體,通過間接ELISA鑒定其亞型。單克隆抗體疊加實驗分析不同單克隆抗體組合的配對效果。溶血抑制實驗鑒定單抗克隆抗體能否抑制LLO的溶血活性。間接ELISA檢測單克隆抗體與威爾斯李斯特菌、英諾克李斯特菌、格氏李斯特菌的交叉性。 結(jié)果：原核表達(dá)出58KD的溶血素蛋白,其序列與Gene Bank公布的序列有99%的同源性。其最優(yōu)化的表達(dá)條件是28℃下用0.1 mmol/L IPTG誘導(dǎo)6 h。。溶血實驗表明重組表達(dá)的LLO具有較強的溶血活性�？偣搏@得13株單克隆抗體,4株為IgGl亞型,9株IgM亞型。溶血抑制試驗表明13株單抗均能不同程度地抑制LLO的溶血反應(yīng)。只有3B4C6、3B4F6與威爾斯李斯特菌、英諾克李斯特菌、格氏李斯特菌有交叉反應(yīng),其余11株均無交叉反應(yīng)。 結(jié)論：成功原核表達(dá)單增李斯特菌的溶血素蛋白(LLO),獲得具有溶血性的LLO。成功制備了抗LLO的單抗克隆抗體,獲得具有單增李斯特菌特異性的單抗。為建立單增李斯特菌特異性的免疫學(xué)檢測方法奠定了基礎(chǔ)。
[Abstract]:Objective to clone hly and express LLO protein and prepare monoclonal antibody against Listeria monocytogenes specific to Listeria monocytogenes (Listeria monocytogenes), which lays a foundation for the establishment of a specific detection method for Listeria monocytogenes (Listeria monocytogenes). Methods: using the DNA of Listeria monocytogenes as template, the hly gene was amplified by PCR and ligated with PET28a () prokaryotic expression vector. After sequencing, it was transformed into E. coli BL21, and induced to express. After optimized expression conditions, a large number of induced expression, purified by nickel column. The expressed protein was identified by SDS-PAGE and westernblotting, and its hemolytic activity was determined by hemolysis test. Balb/c mice were immunized with purified expression products. Monoclonal antibodies against LLO were prepared by hybridoma technique and their subtypes were identified by indirect ELISA. The pairing effect of different monoclonal antibody combinations was analyzed by monoclonal antibody superposition experiment. Hemolytic inhibition assay was used to determine whether monoclonal antibody could inhibit the hemolytic activity of LLO. Indirect ELISA was used to detect the cross-crossing of monoclonal antibodies with Listeria Welsh, Listeria Innoxa and Listeria gravis. Results: 58KD hemolysin protein was expressed in prokaryotic cells, and its sequence was 99% homology with that published by Gene Bank. The optimal expression conditions were induced with 0.1 mmol/L IPTG at 28 鈩，

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