Göbel T W, Meier E L, Du Pasquier L
Institute for Animal Physiology, Munich, Germany.
Eur J Immunol. 2000 Oct;30(10):2775-81. doi: 10.1002/1521-4141(200010)30:10<2775::AID-IMMU2775>3.0.CO;2-U.
The TCR/CD3 complex of a cold-blooded vertebrate, the amphibian Xenopus laevis, was biochemically characterized with a cross-reactive polyclonal antiserum recognizing a conserved epitope in the cytoplasmic domain of CD3E. The specificity and utility of this reagent was validated by Western blot analysis and immunoprecipitation of the well-characterized chicken TCR/CD3 complex. Cross-reactivity with the X. laevis CD3E protein was demonstrated by specific staining of sorted CD8+ cells. Immunohistology on both tadpoles and adult tissues suggests this antiserum will be instrumental in the localization of Xenopus T cells and most likely NK cells. Double staining of tissue sections with an anti-CD8 monoclonal antibody confirmed that this staining is specific. The antiserum was also used for the biochemical analyses of X. laevis TCR/CD3 complex. The 75-kDa alphabeta TCR heterodimer could be separated into a 40-kDa acidic TCR alpha chain and a 35-kDa basic TCR beta chain. Two CD3 proteins, both comigrating at approximately 19 kDa, were associated with the TCR heterodimer. Removal of N-linked carbohydrates yielded CD3 proteins of 19 kDa and 16.5 kDa, most likely representing the CD3epsilon and CD3gamma/delta homologues, respectively. An additional band of 110 kDa represents a multimeric complex of the TCR heterodimer covalently linked to a CD3 dimer. These properties of the Xenopus TCR/CD3 complex substantiate a stepwise evolutionary model for the CD3 protein family.
利用一种能识别CD3E胞质结构域中保守表位的交叉反应性多克隆抗血清，对冷血脊椎动物——两栖动物非洲爪蟾的TCR/CD3复合物进行了生化特性分析。通过蛋白质免疫印迹分析以及对特征明确的鸡TCR/CD3复合物进行免疫沉淀，验证了该试剂的特异性和实用性。通过对分选的CD8⁺细胞进行特异性染色，证明了其与非洲爪蟾CD3E蛋白的交叉反应性。对蝌蚪和成体组织进行的免疫组织学研究表明，这种抗血清将有助于定位非洲爪蟾的T细胞，很可能还有NK细胞。用抗CD8单克隆抗体对组织切片进行双重染色，证实了这种染色具有特异性。该抗血清还用于非洲爪蟾TCR/CD3复合物的生化分析。75 kDa的αβ TCR异二聚体可分离为40 kDa的酸性TCR α链和35 kDa的碱性TCR β链。两种CD3蛋白，均在约19 kDa处共迁移，与TCR异二聚体相关。去除N-连接的碳水化合物后，得到19 kDa和16.5 kDa的CD3蛋白，最有可能分别代表CD3ε和CD3γ/δ同源物。一条额外的110 kDa条带代表TCR异二聚体与CD3二聚体共价连接的多聚体复合物。非洲爪蟾TCR/CD3复合物的这些特性证实了CD3蛋白家族的逐步进化模型。
