Group A Streptococcal Cell Extraction And Purification Of The Active Substance, Its Structure And Activity Analysis

Group A Streptococcus, is a specie of Streptococcus pyogenes in Streptococcus (Streptococcus pyogenes). After penicillin treatment it can be made into group A streptococcus preparation, also known as OK-432. The main clinical use is as a biological response modifier (BRM) by activating the body's immune system involving in tumor combination therapy.Many years of clinical use at home and abroad indicates that the role of OK-432 is activating the immune response, so it is applicable in nearly all tumors, particularly has more pronounced effect on tumors of digestive system, respiratory system cancer, gynecological cancer, hematopoietic malignancies.However until now, functional components and anti-tumor mechanism of Group A Streptococcus have not been clear yet. Which seriously affected clinical application of Group A streptococcus preparation. This article uses modern analytical testing methods, analysis ingredients and chemical composition of Group A Streptococcus; extraced and separated the active ingredient of Group A streptococcus. The purified materials were identified in purity, and its structure, chemical properties and in vitro antitumor activity were also studied. Experimental results are as follows:The first part:Analysis and detection of the major components in intracellular Group A Streptococcus. In this paper, the nutrient composition and contents of Group A streptococcus were systematically studied. Results showed that, the main component is lipoteichoic acid, in which phosphorus content is 24.41 mg/mL. Although there were protein and polysaccharide, molecular weight of the protein after purification was determined with 25～33KD. However, contents of protein and polysaccharide were very low with only 0.057mg/mL and 10mg/mL. Therefore, lipoteichoic acid is the main component of intracellular Group A streptococcus.The second part:Extraction and purification lipoteichoic acid from Group A streptococcus. In this paper, using ion-exchange chromatography (DEAE-52) and gel column chromatography (Sepharose-CL-4B) purified them, and then identified their purity by UV-full wavelength scans and analysis their antibacterial activities. Results showed that:Using anion-exchange chromatography method has a better separation performance, UV scanning revealed no significant protein and nucleic acid contamination. Both LTA extractions showed good antibacterial activity on E. coli and Staphylococcus aureus and had no significant difference. However, Sepharose-CL-4B hydrophobic chromatography does not depend on the negative charge or a specific combination of oligosaccharide structure, and therefore applies more broadly.The third part:Structure and activity analysis of lipoteichoic acid. This paper mainly took structural analysis and activity studies on LTA2 purified by Sepharose-CL-4B hydrophobic chromatography. Result showed that:LTA2 has a similar structure with the standard product LTA. A series of component analysis on LTA2 determined that extraction rate of LTA2 was about 15%. Therefore, phenol extraction and gel chromatography can get the higher purity lipoteichoic acid. Using Gas Chromatography to detect fatty acid and monosaccharide composition of Group A streptococcal lipoteichoic acid. Result showed LTA2 contains five kinds of fatty acids, its main fatty acids are C12:0, C6:0 and C8:0; it only contained glucose.Using MTT method assayed inhibition activities of different concentrations of lipoteichoic acid on Hela cervical cancer cells at different time points in vivo. Activity tests proved that inhibition activities of LTA2 on Hela cells in vitro were significantly different; lipoteichoic acid can inhibit the proliferation of Hela cells in a certain dose range.