猪CD4分子的全长cDNA克隆与序列分析

Sequence Analysis and Cloning of Porcine Full-length CD4 cDNA

CD 4分子为动物辅助性T细胞(TH)和部分胸腺细胞的共受体与信号传导分子,参与TCR介导的TH细胞活化和胸腺细胞分化过程。本研究应用RT-PCR和RACE技术从猪胸腺细胞总RNA中扩增克隆了猪CD 4全长cDNA序列,并进行了序列特性分析。序列分析结果表明,在猪胸腺细胞和外周血淋巴细胞中存在2种方式剪接的CD 4 mRNA转录本,其中一种mRNA转录本缺失编码40个氨基酸残基的120 n t序列,提示该转录本可能编码猪分泌型CD 4分子;猪CD 4全长cDNA序列为2 715 n t,其中5′非编码区159 n t,3′非编码区1 183 n t,1 374 n t的开放阅读框编码457个氨基酸的猪CD 4前体蛋白(含4个糖基化位点);猪CD 4分子的7个Cys残基(Cys43、Cys122、Cys327、Cys418、Cys421、Cys444和Cys446)和2个Ser残基(Ser432和Ser439)在动物种间保守;在猪CD 4分子胞浆区,存在高度保守的S rc家族蛋白酪氨酸激酶p56lck识别位点KKTCQC和内化相关双亮氨酸基序。推导氨基酸序列分析结果显示,猪与人、兔、猫、狗和鼠CD 4蛋白的氨基酸同源性分别为56.0%,54.5%,56.9%,56.5%和44.9%。

As an important co-receptor and signal molecule expressed on surface of T cells and thymocytes ,CD4 is involved in the activation of peripheral T help cells and differentiation of thymocytes. The porcine CD4 cDNA was amplified and cloned from total RNA of lymphocytes and peripheral blood lymphocytes by RT-PCR and RACE（rapid amplification of cDNA ends）. Sequence analysis revealed that two alternatively spliced transcripts of porcine CI）4 mRNA were in the lymphocytes and peripheral blood lymphocytes,and the shorter transcripts is devoid of 120 nt encoding 40 amino acid residues,suggesting that it may encod a porcine secreted CD4 protein;Amplified porcine CD4 full-length cDNA is 2715 nt in length,including 159 nt of 5＇ untranslated region, 1374 nt of coding region and 1183 nt of 3＇ un-translated region. The coding region encoded a protein precursor with 457 amino acid residues and four N-glycosylation site （Asn-X-Ser or Asn-X-Thr）; Seven cysteine residues （Cys^43. Cys1^21.Cys^327. Cys^416, Cys^421, Cys^444 and Cys^446 and two serine residues （Ser^431 and Ser^438） are conserved among mammalian species ;Additionally,The porcine CD4 contains highly conserved protein tyrosine kinase p56^kk recognition site KKTCQC and a di-leucine motif involved in Ag-induced CD4 internalization within the cytoplasmic domain. Comparison of the deduced amino acids sequence of porcine CD4 with those of human,rabbit,cat,dog and mouse showed that the amino acids homology were 56.0%,54.5%,56.9%,56.5%and 44.9%,respectively.