The Study On The Extractionand Purification Technology Of The Lipopolysaccharideantigen Of Inactivated Mycobacterium Tuberculosis

The Lipopolysaccharides antigen of Mycobacterium tuberculosis is an effectiveantigen,and it is widely used. Its main constituent is LAM,which comes from LPS.Therefore，this thesis studies the extraction，separation and purification technology ofMycobacterium tuberculosis in order to find the optimal separation and purificationprocess route, so as to improve the yield of LAM.1The extraction of LPSFirst, taking the single-factor test for each of the factors that may affect the rateof cell disruption.Then,according to the measured yield of LPS，we can determinethe factors and their respective level that will be participated inthe next orthogonal experimental design. Different from other literatures, this topicCombined with grinding, ultrasonication and ethanol backflow for the test.At the completion of the orthogonal test, the optimal program as follows：the brokenpower is650w, the reaction time is1h and the ethanol concentration is50%，brokennumber is50，twice extracted.2The technology of DeproteinizationProtein residues will affect the quality of the final rate of LAM,so it is necessaryto remove protein from the extraction.The experiment choosed the hot phenol watermethod and Sevag method to remove protein by single-factor test and orthogonalexperiment to establish a better process.Considering the retention rate ofpolysaccharide and the removal rate of protein integratedly,the effect of hot phenolmethod of removing protein was better, and then optimized its process. the optimal solutions were obtained as follows： phenol concentration of55%, theoscillation time of1h, centrifuged for30min.3The purification of LipopolysaccharideAfter extraction and separation，the sample solution should be skimmed andpurified with column chromatography, with anthrone-sulfuric acid method trackingeluents for the sugar concentration.And then detecting the enzyme values(OD₄₅₀).Eventually the yield of LAM is6.84mg/g.The results show that the yield of LAM can be greatly improved by optimizationof the extraction,separation and purification of LPS.