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
a College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Key Laboratory of Animal Production and Product Quality Safety of Ministry of Education , Jilin Agricultural University , Changchun , China.
Biosci Biotechnol Biochem. 2017 Aug;81(8):1489-1496. doi: 10.1080/09168451.2017.1340086. Epub 2017 Jun 23.
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.
重组激活基因2（RAG2）对于未成熟B细胞分化是必需的。目前市面上有针对人和兔RAG2的抗体，但针对猪RAG的抗体至今仍未可得。在本研究中，合成了猪RAG2基因序列，然后将其克隆到pET-28a载体中。重组融合蛋白在大肠杆菌中成功表达，通过镍柱层析纯化，并进一步用烟草蚀纹病毒蛋白酶消化。经切割的蛋白通过分子筛层析纯化并命名为pRAG2。我们用pRAG2免疫兔子，收集血清并纯化兔抗pRAG2多克隆抗体。兔抗pRAG2多克隆抗体通过免疫荧光在过表达pRAG2的真核细胞上进行检测，并且在蛋白质印迹中也能够识别猪天然RAG2和人RAG2蛋白。这些结果表明，所制备的兔抗pRAG2多克隆抗体可作为检测猪未成熟B细胞分化的工具。
