1. Expression and purification of swine RAG2 in E. coli for production of porcine RAG2 polyclonal antibodies.
- Author
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Jin YB, Yang WT, Huang KY, Chen HL, Shonyela SM, Liu J, Liu Q, Feng B, Zhou Y, Zhi SL, Jiang YL, Wang JZ, Huang HB, Shi CW, Yang GL, and Wang CF
- Subjects
- Animals, Antibodies isolation & purification, Antibodies metabolism, Blotting, Western, Cloning, Molecular, DNA-Binding Proteins genetics, DNA-Binding Proteins immunology, Endopeptidases chemistry, Escherichia coli metabolism, Fluorescent Antibody Technique, Genetic Vectors chemistry, Genetic Vectors metabolism, Humans, Immune Sera chemistry, Isoenzymes biosynthesis, Isoenzymes genetics, Isoenzymes immunology, Nuclear Proteins genetics, Nuclear Proteins immunology, Rabbits, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Swine, VDJ Recombinases genetics, VDJ Recombinases immunology, Antibodies chemistry, DNA-Binding Proteins biosynthesis, Escherichia coli genetics, Gene Expression, Nuclear Proteins biosynthesis, VDJ Recombinases biosynthesis
- Abstract
Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2. We used pRAG2 to immunize rabbits, collected the serum and purified rabbit anti-pRAG2 polyclonal antibodies. The rabbit anti-pRAG2 polyclonal antibodies were tested via immunofluorescence on eukaryotic cells overexpressing pRAG2 and also able to recognize pig natural RAG2 and human RAG2 protein in western blotting. These results indicated that the prepared rabbit anti-pRAG2 polyclonal antibodies may serve as a tool to detect immature B cell differentiation of swine.
- Published
- 2017
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