Cloning, Sequence Analysis And Tissue Expression Of Gost CD4 Gene CDNA

CD4 molecule is one of the cell-surface glycoprotein family and is most well characterized on the surface of thymocytes and a subset of mature T cells. As an important co-receptor and signal moleculer expressed on surface the major histocompatibilitycomplex classâ…¡(MHCâ…¡)molecule of T cells and thymocytes, CD4 is involved in the activation of peripheral T help cells and differentiation of thymocytes. CD4 also serves as the primary cellular receptor for human immunodeficiency virus 1 (HIV-1).Based on the CD4 mRNA and EST sequences of sheep, and the mRNA sequences of the CD4 molecules from the species reported or submitted to Genbank, the special primer of goat has been designed. The full-length cDNA of goat was amplificated by the RT-PCR technique using the total RNA isolated from goat testis as template. Then its potential amino acid sequence was deduced and its protein structure was analysed. Meanwhile, the tissue expression from different goat tissues was studied by RT-PCR and Fluorescence quantitative PCR methods. The main results are as following:1. The full-length cDNA of goat CD4 gene was obtained and deposited under the GenBank accession number EU913093. Its length was 1555nt, including a sequence of 149bp as 5'-untranslated region (5'UTR). It encoded a polypeptide of 455 amino acids, with 50.50kD molecular weight and 9.52 isoelectric point.2. The analysis of amino acids showed that the goat CD4 molecule was a cell-surface glycoprotein. It covered the signal peptide, extracellular region, transmembrane domain and cytoplasmic domain, with 25, 352, 23 and 36 acidic amino acids, respectively. The extracellular region contains four immunoglobulin-like domains, two disulfide bonds (C⁴¹-C¹⁰⁹ and C¹⁴³-C¹⁸⁰) and three N-glycosylation sites (N²³¹, N²⁶³, N³⁴³) . Comparison of amino acids sequence of goat CD4 with those of sheep(Ovis aries), pig(Sus domestica), horse(Equus caballus), human( Homo sapiens sapiens), rabbit(Oryctolagus cuniculus), dog(Canis familiaris), cat(Felis catus), mouse(Mus musculus) and bat(Rousettus aegyptiacus), the similarity were 98%, 81%, 72%, 70%, 75%, 74%, 73%, 66% and 70%, respectively. It showed that it had the highest amino acids homology with that of sheep.3. The tissue expression analysis indicated that the goat CD4 was expressed in thymus, spleen ,ovary , lymph, pancreas ,testis and muscle. Real time quantitative PCR showed that CD4 mRNA was highly expressed in the lymph and immue function.