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- Title
Expression and purification of swine RAG2 in E. coli for production of porcine RAG2 polyclonal antibodies.
- Authors
Jin, Yu-Bei; Yang, Wen-Tao; Huang, Ke-Yan; Chen, Hong-Liang; Shonyela, Seria-Masole; Liu, Jing; Liu, Qiong; Feng, Bo; Zhou, You; Zhi, Shu-Li; Jiang, Yan-Long; Wang, Jian-Zhong; Huang, Hai-Bin; Shi, Chun-Wei; Yang, Gui-Lian; Wang, Chun-Feng
- 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 swineRAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed inE. 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.
- Subjects
RECOMBINATION activating genes; CLONING; ESCHERICHIA coli
- Publication
Bioscience, Biotechnology & Biochemistry, 2017, Vol 81, Issue 8, p1489
- ISSN
0916-8451
- Publication type
Article
- DOI
10.1080/09168451.2017.1340086