论文部分内容阅读
目的对以ＧＳＴ－ｇｐ１３０融合蛋白为探针，筛选人胎盘ｃＤＮＡ文库而得到的一个分子（ＧＡＭ，ｇｐ１３０ａｓｓｏｃｉａｔｅｄｍｏｌｅｃｕｌｅ）ｃＤＮＡ序列进行测定并做真核表达。方法进行噬菌体制备及克隆测序，并做同源性检索和构型、亲水性分析，ＣＯＳ细胞转染和３５Ｓ－蛋氨酸标记。结果所得ＧＡＭ分子与另一已克隆成功但功能还不清楚的称为ｈＡＥＳ－１的分子同源性达９７．７％。此外，ＧＡＭｃＤＮＡ还与果蝇ｅｎｈａｎｃｅｒｏｆｓｐｌｉｔ基因相关的分子有较高同源性。ＧＡＭ分子具有一个５８８个核苷酸的开放读框，编码１９６个氨基酸。将ＧＡＭ分子ｃＤＮＡ转染ＣＯＳ细胞后，细胞裂解液电泳可见一分子量约为２４×１０３的表达带，与预期的分子量相符合。结论ＧＡＭ分子与ｈＡＥＳ－１分子在开放读框内仅相差一个氨基酸（且不排除测序的个别错误），其分子量也与已报道的ｈＡＥＳ－１分子量相同，故认为两者很可能是同一分子。
Objective To determine the cDNA sequence of a molecule (GAM, gp130associatedmolecule) obtained by screening the human placenta cDNA library with GST-gp130 fusion protein as a probe and make eukaryotic expression. Methods Bacteriophage preparation and cloning and sequencing were performed. Homology search and conformation, hydrophilicity analysis, COS cell transfection and 35S-methionine labeling were performed. Results The resulting molecule of GAM had 97.7% homology to another molecule known as hAES-1, which had been cloned but functionally unclear. In addition, GAMcDNA also has high homology to Drosophila enhancerofsplit gene-related molecules. The GAM molecule has a 588 nucleotide open reading frame encoding 196 amino acids. GAM cDNA transfection of COS cells, the cell lysate electrophoresis showed a molecular weight of about 24 × 103 expression bands, consistent with the expected molecular weight. Conclusions GAM molecules and hAES-1 molecules differ by only one amino acid in the open reading frame (and do not rule out sequencing errors), and their molecular weights are also the same as those reported for hAES-1. Therefore, they are likely to be the same molecule.