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CD4 and CD8 Molecules of Non-human Primates; Molecular Cloning and Their Roles in SIV Infection

非人类灵长类动物的 CD4 和 CD8 分子；

基本信息

批准号：
03454109
负责人：
TATSUMI Masashi
金额：
$ 4.16万
依托单位：
National Institute of Health (N.I.H.)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454109/
关键词：
Cynomolgus monkey CD4 CD8 Molecular cloning Chimeric CD4 in vitro expression SIV HIV 形質転換細胞 CD4遺伝子、CDRー1領域、PCR,アミノ酸置換 cDNA 塩基配列決定

项目摘要

To elucidate the interaction between SIV and monkey CD4 and CD8 molecules, we attempted molecular cloning and to compare those with human counterparts. Cynomolgus monkey thymocyte cDNA library was constructed in a lambdaZAP phage vector system by inserting cDNA synthesized from thymocyte mRNA with Gubler-Hoffmann method, and screened by plaque hybridization with PCR-generated putative monkey CD4 and CD8 gene fragments. The coding region of monkey CD4 gene was revealed to consist of 1377 nucleoside acids and show high homology (95%) to human CD4 gene. Alignments of deduced amino acid sequences revealed that only one amino acid was substituted in both signal peptide and transmembrane domains, and that there was no difference in its cytoplasmic domain. The substitution of amino acids was concentrated on CDR-1 region of extracytoplasmic Ig-like V1 domain, suggesting the possible conformational change in this region reported to be important for HIV binding. We constructed mammalian expression vectors with monkey, human and their chimeric CD4 genes and established Hela transformants expressing respective CD molecules on the cell surface. The susceptibility of these cells to HIV and SIV was examined by synthcium formation and by detection of provirus with PCR method. HIV could infect not only transformant expressing human V1 domain but also monkey V1 domain suggesting that monkey CD4 molecule might serve as the cellular receptor to both SIV and HIV.The coding region of monkey CD8 gene was revealed to consist of 708 residues and also show high homology (95%) to human CD8. Alignments of deduced amino acid sequences displayed that the substitution was distributed evenly in the entire coding region, differing from monkey CD4. Expected 26KDa molecule was produced by in vitro translation method with monkey CD8 gene.

为了阐明SIV与猴CD 4和CD 8分子之间的相互作用，我们尝试了分子克隆并与人类对应物进行比较。以Gubler-Hoffmann法合成的胸腺细胞cDNA为模板，构建了食蟹猴胸腺细胞cDNA文库，并与PCR扩增的猴CD 4和CD 8基因片段进行噬斑杂交筛选。猴CD 4基因编码区由1377个核苷酸组成，与人CD 4基因同源性高达95%。推导的氨基酸序列的比对显示，只有一个氨基酸被取代的信号肽和跨膜结构域，并在其胞质结构域没有差异。氨基酸取代集中在胞质外Ig样V1结构域的CDR-1区，表明该区域可能的构象变化对HIV结合很重要。我们构建了含有猴、人及其嵌合CD 4基因的哺乳动物表达载体，并建立了在细胞表面表达相应CD分子的Hela转化子。用PCR方法检测前病毒的表达，并观察细胞对HIV和SIV的易感性。猴CD 4分子可能是SIV和HIV的细胞受体。猴CD 8基因编码区由708个残基组成，与人CD 8基因有95%的同源性。推导的氨基酸序列的比对显示，取代均匀分布在整个编码区，不同于猴CD 4。利用猴CD 8基因通过体外翻译方法产生了预期的26 KDa分子。