Recombinant Streptolysin O: A Versatile Toxin with Wide Applications？ Introduction Recombinant Streptolysin O (Slo) is a toxin produced by GAS that can create large pores in cell membranes, allowing toxic proteins to enter the cytoplasm and exert their harmful effects. It is also highly antigenic, with the majority of patients infected with streptococci developing Slo antibodies. The measurement of anti-streptolysin O (ASO) antibodies in serum has widespread applications in the diagnosis of diseases related to streptococcal infections such as glomerulonephritis, scarlet fever, tonsillitis, rheumatic fever, and reactive arthritis. The development of effective detection methods for Slo antibodies in patient serum is of great value in the identification and evaluation of these diseases. Applications Recombinant Streptolysin O has a wide range of potential applications. It can be used for the detection of ASO in diseases related to streptococcal infections and reactive arthritis. Additionally, it can enhance cell membrane permeability, allowing the entry of large molecular proteins and nucleotides into recipient cells for reprogramming purposes. This strategy provides a new source of seed cells for the development of artificial organs and offers a novel approach for the replacement and repair of damaged tissue and organs. Furthermore, Recombinant Streptolysin O can be used as an adjunctive therapy for streptococcal pneumonia. Currently, Slo can be obtained through natural cultivation or recombinant expression. While natural cultivation allows for the production of Slo with a native structure, it is complex and yields are low. This study lays the foundation for the large-scale production of small molecular Slo antigens with both hemolytic and specific immunological activities using the E. coli expression system. Induction of Recombinant Streptolysin O Expression The engineered gene bacteria were inoculated into LB medium containing ampicillin and incubated overnight at 37°C with shaking. The culture was then transferred to fresh LB medium and incubated at 37°C with shaking until the OD600 value reached approximately 0.5. At this point, IPTG was added to a final concentration of 1mmol/L to induce expression for 4 hours. The cells were then centrifuged, and the bacterial pellets were collected for SDS-PAGE analysis. Purification of Recombinant Streptolysin O The bacterial pellets were collected by centrifugation and washed three times. The pellets were resuspended in E. coli sonication buffer and subjected to sonication. The inclusion bodies were collected by centrifugation, washed with 2M urea, and dissolved in a denaturing solution. The recombinant protein was then purified using Ni-NTA agarose chromatography according to the manufacturer's instructions. The purified protein was identified by 12% SDS-PAGE. The denatured protein solution was gradually added dropwise to a refolding solution with continuous stirring. After a 20-fold dilution, the mixture was incubated at 4°C for 24 hours for refolding. The protein solution was then concentrated using a Millipore ultrafiltration tube for further use. References [1] Yang S, Pan J. Preparation of recombinant Streptolysin O with active structure of pyogenic streptococcus. [2] Sun H, Zhang Z. Expression, purification, and identification of the biological activity of human hydrogen peroxidase in E. coli. Journal of Jinan University, 2014, 35(2): 187-191. 查看更多
磁珠在生物分离和富集中的应用？ 作为一种新型纳米材料，磁珠具有广泛的应用前景。它可以用于检测毒素、病毒、细菌等，也可用于分离特定生物分子。磁性分离技术利用磁性微粒表面的配体和受体间的特异性亲和作用，通过外加磁场的定向控制，可以从复杂的原始生物体系中直接分离出目标生物分子。这种技术具有简单方便和高选择性的双重优势，为生物分子的快速分离和富集提供了一种强有力的手段。 磁珠的制备方法 根据Liu等人的研究，可以使用NHS/EDC化学偶联方法将单克隆抗体与磁珠化学偶联。 首先，用50 mM MEST溶液（含0.05% Tween-20，pH 5.2）洗涤磁珠，然后加入EDC和NHS反应。接着用MEST溶液和BST溶液洗涤磁珠，并加入2A7H6抗体进行反应。最后，将免疫磁珠保存在含NaN3和BSA的BST溶液中。 Mag-Beads His-Tag蛋白纯化磁珠 首先，用PBS溶液洗涤免疫磁珠，然后加入Tm富集液进行反应。接着用PBS溶液洗涤磁珠，并用甘氨酸-HCl缓冲液洗脱蛋白。最后，将洗脱后的免疫磁珠保存在适当的缓冲液中。 参考文献 [1] 李文娇. 不同磁珠对过敏原蛋白纯化效果的比较研究. [2] Liu Y, Zhang Z, Wang Y, et al. A highly sensitive and flexible magnetic nanoprobe labeled immunochromatographic assay platform for pathogen Vibrio parahaemolyticus[J].查看更多
小鼠肠微血管细胞试剂盒的应用及其对小鼠原位结肠癌生长的影响？ 背景： [1-6] 小鼠肠微血管细胞试剂盒是一种用于培养小鼠肠微血管细胞的试剂盒。该试剂盒包含多种组分，如组织解离液、组织处理缓冲液、成纤维抑制剂、组织洗液、生长因子及血清、基础培养基和预备液。 微血管是心血管系统中的微细血管，只能在显微镜下观察到。它们是连接小动脉和小静脉的细小血管，分布于各种组织和器官中，形成一个网状结构，也被称为终末血管床。小鼠肠微血管细胞可以保持原始细胞的分化状态，可用于评估体外药物模型系统和调节特定基因的遗传功能。通过使用小鼠肠微血管细胞试剂盒中提供的肠微血管组织分离体系，可以分离出肠微血管细胞，尽管肠微血管组织具有很强的机械韧性，但经过EDTA/EGTA处理的肠微血管组织会改变微血管细胞的某些功能特性。 应用： [7][8] 巨噬细胞金属弹力酶基因转染CT-26细胞对小鼠原位结肠癌生长及微血管生成的影响 巨噬细胞金属弹力酶（MME）是基质金属蛋白酶（MMP）家族的一员，也被称为MMP-12。与其他MMP成员不同，MME能够分解纤溶酶原，产生具有抑制血管内皮细胞增殖作用的血管抑素（angiostatin），从而抑制肿瘤细胞的生长，在抗肿瘤血管生成中起重要作用。本研究通过PCR扩增编码MME基因结构域Ⅰ和Ⅱ的cDNA片段，并将其克隆入真核细胞表达载体中，然后将构建的真核细胞表达载体转染到小鼠CT-26结肠癌细胞中。通过RT-PCR、免疫细胞化学和Western blot等方法，鉴定MME mRNA和重组蛋白在CT-26细胞中的表达。 通过体外分解胶原蛋白和明胶酶谱方法，鉴定了MME重组蛋白的酶活性。建立了MME转染组和对照组的小鼠原位结肠癌种植模型，观察了MME对原发性结肠癌生长的影响，并通过免疫组织化学、原位杂交和Western blot等方法检测了肿瘤组织中的微血管密度和VEGF的表达。 参考文献 [1] Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair. Madlener M, Parks WC and Werner S. Experimental Cell Research. 1998. [2] Mouse macrophage metalloelastase gene transfer into a murine melanoma suppresses primary tumor growth by halting angiogenesis. Gorrin-Rivas MJ, Arii S, Furutani M et al. Clinical Cancer Research. 2000. [3] Emphysema and metalloelastase expression in mouse lung induced by cigarette smoke. Valenca SS, da Hora K, Castro P et al. Toxicologic Pathology. 2004. [4] Macrophage metalloelastase, MMP-12, cleaves human apolipoprotein(a) in the linker region between kringles IV-4 and IV-5. Edelstein C, Shapiro SD, Klezovitch 0 et al. Journal of Biological Chemistry. 1999. [5] Hyaluronan and type III pro-collagen peptide concentrations in bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis. Bjermer L, Lundgren R and Hallgren R. Thorax. 1989. [6] Macrophage metalloelastase as a major factor for glomerular injury in anti-glomerular basement membrane nephritis. Kaneko Y, Sakatsume M, Xie Y et al. J Immunol. 2003. [7] Lung morphometry and MMP-12 expression in rats treated with intraperitoneal nicotine. Valenca SS, de Souza da Fonseca A, da Hora K et al. Experimental and Toxicologic Pathology. 2004. [8] 石海.巨噬细胞金属弹力酶基因转染CT-26细胞对小鼠原位结肠癌生长及微血管生成的影响[D].安徽医科大学,2005. 查看更多
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