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Expression of the NS3 protein of hepatitis C virus used in the HCV UMELISA® test kit


Journal of Bacteriology & Mycology: Open Access
José Miguel Fernández Torres,1 Yeosvany Cabrera Artiles,1 María Teresa Barcelló Ávila,1 Ronny Camacho Pupo,2 Duniesky Martínez García,1 Alina Sobrino León1

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Abstract

To manufacturing the HCV UMELISA® Test kit, a recombinant NS3 protein is used as part of the antigen mixture. This protein is obtained from Escherichia coli XL-1Blue transformed with pR2M6-NS3-His plasmid downstream trp promoter. Regulation of expression has been identified as the main cause that affects yield and reproducibility at the fermentation process. To improve protein expression and transcription regulation, cloning of NS3 sequence was performed downstream tac promoter. At the same time, three E. coli strains (XL 1-Blue, W3110 and C600) were used to assess the expression of the NS3 protein. The tac promoter improved the expression level at all the strains evaluated from 20% under trp until 30 % and a reduction of promoter leakage before induction was observed compared with trp promoter. Due to the insensibility to the high metabolic burden, C600 was settled as the strain to use for NS3 expression. The new construction was well recognized by a pool of positive sera and individual sera from infected humans in an indirect ELISA using the HCV UMELISA® Test kit. 

Keywords

HCV, C virus, ELISA, UMELISA, E. coli, hepatocellular, carcinoma, antibodies detection, nucleocapsid, NS3-tac protein, mutation, promoter, strain, diagnostic test

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