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
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