猪CD3∈分子的cDNA克隆与序列分析

Cloning and sequence analysis of porcine CD3∈ cDNA

应用RT-PCR和3′RACE技术,本研究从猪外周血淋巴细胞总RNA中扩增克隆了猪CD3ε基因并进行了序列分析。序列分析结果表明,猪CD3εcDNA序列长1241nt,其中5′非翻译区80nt,3′非翻译区570nt,开放阅读框591nt,该开放阅读框编码196个氨基酸残基的CD3ε前体蛋白(无糖基化位点);在猪CD3ε蛋白的胞外区,Cys49、Cys91、Cys108和Cys111为保守性氨基酸残基,它们与CD3ε免疫球蛋白样结构的形成有关。在猪CD3ε蛋白胞浆区,存在免疫受体酪氨酸活化基序(ITAM)、内质网潴留基序和1个SH3结合基序,其中ITAM基序含有2个SH2结合基序和2个潜在酪氨酸磷酸化位点Tyr177和Tyr188,这些基序是猪CD3分子参与T细胞活化信号转导和TCR-CD3复合体装配的结构基础。推导氨基酸序列分析结果表明,猪与人、鼠、狗、牛和绵羊CD3ε蛋白的氨基酸同源性分别为61.2%、58.7%、58.7%、65.1%和65.6%。

The porcine CD3ε cDNA was amplified and cloned from total RNA of peripheral blood lymphocytes cultured under stimulation of concanavalin A (Con A) by RT-PCR and 3′RACE (rapid amplification of cDNA ends). Sequence analysis revealed that the amplified porcine CD3ε cDNA was 1 241 nt in length, including 80nt of 5′un-translated region,591 nt of coding region and 570 nt of 3′un-translated region. The coding region encoded a predicted molecular mass of 22 kD protein precursor with 196 amino acids residues lacking N-glycosylation site. The porcine CD3ε contained several characteristic motifs highly conserved across the species within the cytoplasmic domain, namely Src homology 3(SH3),endoplasmic reticulum (ER) retention motif and the immunoreceptor tyrosine-based activation motif(ITAM) which consisted of two Src homology 2(SH2) and two potential tyrosine phosphorylation sites at Tyr 177 and Tyr 188. These motifs were associated with signal transduction on T-cell activation as well as assembly and expression of TCR-CD3 complex. Comparison of the deduced amino acids sequence of porcine CD3ε with those of human,mouse,dog,bovine and sheep showed that the amino acids homology were 61.2%, 58.7%,58.7%,65.1% and 65.6%, respectively.