人血小板生成素（hTPO）的cDNA克隆与序列测定

cDNA Cloning and Sequencing of Human Thrombopoietin

由于人血小板生成素（ｈＴＰＯ）ｃＤＮＡ的阅读框架较长（１０５９ｂＰ）而且序列中无合适的酶切位点，因此，我们利用一个同义突变创造的ＫｐｎＩ酶切位点人为将ｈＴＰＯｃＤＮＡ分成Ｎ－端和Ｃ－端两个片段。首先利用反转录聚合酶链式反应（ＲＴ－ＰＣＲ）从人胎肝ｍＲＮＡ中分别扩增出两个ｃＤＮＡ片段，于ＥｃｏＲＩ、ＫＰｎＩ位点分别克隆入测序载体ｐＵＣ１９中。序列测定表明，克隆片段与国外已报道的ｈＴＰＯｃＤＮＡ序列完全一致。

A Synonymous Substitution was used to set up a novel restriction enzyme site-KpnI,separating the total cDNA into two fragments,encoding N terminal and C terminal,respectively.The fragments were amplified from human fetalliver mRNA,coined into pUC 19 and sequenced.Their sequences were shown to be identical with hTPO cDNA reported.