ABSTRACT
OBJECTIVE: To express and purify of the BC097361 recombinant protein, and to prepare the BC097361 specific rabbit polyclonal antibody. METHODS: BC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a (+), and the resulting plasmid was transformed into E.coli BL21 (DE3). The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting. The expressed product was purified using Ni+ affinity column chromatography.Then the purified pET-32a (+) -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. RESULTS: The BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody more than 1:320000. The high specificity was confirmed with Western blot. CONCLUSIONS: The recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.
Subject(s)
Antibodies/metabolism , Antibody Specificity , Recombinant Fusion Proteins/biosynthesis , Angiotensin II/genetics , Animals , Antibodies/immunology , Antibodies/isolation & purification , Blotting, Western , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/genetics , Liver Cirrhosis/genetics , Male , Plasmids/genetics , Rabbits , Recombinant Fusion Proteins/immunology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: To detect TB specific T cell responses by using the recombinant ESAT-6 protein as stimulus in Chinese HIV infected patients. METHODS: ELISPOT-IFN-gamma assay by using the recombinant ESAT-6 protein as stimulus to detect specific T cell responses in HIV+ patients with or without clinical manifestation of TB diseases. RESULTS: Recombinant ESAT-6 protein specific T cell responses show significant high frequencies in both of TB patients with or without HIV infection than that in the healthy control and HIV+ group without clinical TB diseases. CONCLUSION: The ELISPOT-IFN-gamma assay by using recombinant ESAT-6 protein as stimulus could be used in diagnoses of TB infection in Chinese HIV infected patients.
