Leptospira is one of the most common zoonotic diseases in the tropics and subtropics. Humans are infected by exposure to Leptospira contained water or food sources. Leptospirosis usually breaks out after the flood and causes several consequences for people and economy. Leptospirosis disease, if not rapidly detected and treated promptly, it causes serious consequences such as acute hepatitis-kidneys, meningitis and bleeding, heart and nerve complications, and severe illness can lead to death. Therefore, quick and accurate detection of Leptospira pathogen plays a very important role in Leptospirosis disease treatment. Among antigens of Leptospira, a conserved domain of LigB antigen (Leptospiral immunoglobulin-like protein) was reported that is present in the most of pathogenic serovars of Leptospira, but not in the non-pathogenic Leptospira biflexa, thus this conserved domain was used for production of Leptospirosis detection kits as well as vaccine for Leptospirosis. In order to create a kit for Leptospirosis diagonostic, especially detect anti-Leptospira antibodies in Leptospira infected serum and plasma samples, about 1kb gene fragment encoding for conserved domain of LigB (about 36 kb in molecular weight) was used as the material for producing of LigB protein by DNA recombinant technology. In this study, we present the results for cloning, expressing a conserved domain of LigB antigen in E. coli cells and purifying protein by affinity chromatography collumn. The result indicates that recombinant LigB protein was successfully expressed in E. coli Rosetta 1 and purified by Hitrap chealating collumn. The LigB protein concentration after purification reached 60 mg/L medium with 98% purity. This purified protein will be used as the materials for creating Leptospirosis kit.
Leptospirosis is a common zoonotic disease in the tropics and subtropics. Leptospira infected human without prompt detection and treatment will face serious consequences such as acute hepatitis-kidneys, pulmonary hemorrhage which can lead to death. Besides the MAT gold standard method, Leptosipra antigens developed by DNA recombination technology have been widely studying and applying in diagnosis of Leptospira infection in human and animal. Overcoming the disadvantages of MAT and ELISA such as complicated protocol, which requires highly qualified staff and specialized equipment, the latex agglutination method has been studied and widely used in detecting pathogens such as Salmonella, E. coli, Leptospira in the world. The advantages of this method are simple operation, fast and cheap. In the previous article, we expressed Leptospira LigB antigen in E. coli cells and successfully purified it by affinity chromatography with 98% purity. In this paper, we present the process for establishment of a Lepto-LAT kit to detect Leptospira infection in dogs. This kit had the sensitivity and specificity of 91.75% and 91.57%, respectively.
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