Sds - page and activity staining analysis revealed that all of clones 2 , 5 and 9 exhibited a single active band with 83 - kd in size , whereas clone 3 exhibited four different bands with 64 kd , 70kd , 76kd and 83 kd after construcing a series of deleted subclones from the plasmid prepared from clone - 5 , a complete nucleotide sequences comprising of 3 , 205 bps wes determined . one open reading frame ( orf ) was found in this sequence , which was comprised of 2 , 661 bps and could encode a polypeptide with 887 amino acid residues 利用基因分析软件genetyx对clone - 5质粒的核苷酸序列进行分析,并搜索日本dna数据库( dnadatabankofjapan ,简称ddbj ) ,推断clone - 5质粒的几丁质酶基因含有一个2 , 261bp的开放阅读框( orf ) ,编码一条887个氨基酸的几丁质酶蛋白; orf上游有一个启动子。
( 3 ) isolation and culture of human primordial germ cells ( pgcs ) . human pgcs collected from gonadal ridges and mesenteries were grown on mouse feeder layers in the presence of human recombinant leukemia inhibitory factor ( lif ) , human recombinant basic fibroblast growth factor , and forskolin as described previously . initially , pgcs were visualized by alkaline phosphatase activity staining ( 3 )人类pgcs的分离和培养从4 10周龄药物流产胚胎的生殖嵴和肠系膜组织中分离原始生殖细胞( primordialgermcells , pgcs ) ,培养在添加人重组白血病抑制因子( lif ) 、人重组碱性成纤维细胞生长因子( bfgf )和forskolin的小鼠饲养层细胞上。