Zhang Xuelian, Wei Shuangshi, Shao Jianwei, Zhang Shudong, Gao Mingchun, Zhang Wenlong, Ma Bo, Wang Junwei
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China.
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China.
Gene. 2015 Jun 15;564(2):160-7. doi: 10.1016/j.gene.2015.03.034. Epub 2015 Mar 18.
CD3 is one of the most important cell surface markers of T lymphocytes which play an important role in signal transmission of antigen recognition. In this study, goose CD3ε gene was cloned by touchdown PCR with the template of goose thymus cDNA. The complete open reading frame of goose CD3ε encoded 178 amino acid residues with a 21 signal peptide. Sequence alignments showed that goose CD3ε had an amino acid sequence similarity to duck (80.3%) and chicken (66.3%). The extracellular domain of goose CD3ε was efficiently expressed as fusion protein in Escherichia coli, purified by a Ni-NTA agarose column, and the purified recombinant protein was used to produce anti-GoCD3εex polyclonal antibodies. The characteristics of PAb were identified by Western blot, cellular ELISA, IFA, FCM, and LSCM analysis. These results may be useful for a better understanding of goose CD3ε and have a foundation for the study of T cell mediated immune mechanism in waterfowl.
CD3是T淋巴细胞最重要的细胞表面标志物之一，在抗原识别信号传导中起重要作用。本研究以鹅胸腺cDNA为模板，通过降落PCR克隆了鹅CD3ε基因。鹅CD3ε的完整开放阅读框编码178个氨基酸残基，带有一个21个氨基酸的信号肽。序列比对显示，鹅CD3ε与鸭（80.3%）和鸡（66.3%）的氨基酸序列具有相似性。鹅CD3ε的胞外结构域在大肠杆菌中高效表达为融合蛋白，经镍-亚氨基二乙酸琼脂糖柱纯化，纯化后的重组蛋白用于制备抗GoCD3εex多克隆抗体。通过蛋白质免疫印迹、细胞酶联免疫吸附测定、间接免疫荧光法、流式细胞术和激光扫描共聚焦显微镜分析鉴定了多克隆抗体的特性。这些结果可能有助于更好地了解鹅CD3ε，并为研究水禽T细胞介导的免疫机制奠定基础。
