人血小板生成因子（TPO）的cDNA克隆及促血小板生成效应

Cloning and expression of human thrombopoietin (TPO)cDNA and stimulative effect of thrombopoietin on platelet production

用ＲＴ－ＰＣＲ方法，从人胎肝总ＲＮＡ中扩增得到约１．１ｋｂ人ＴＰＯｃＤＮＡ编码顺序，并进行分子克隆．将其中一个克隆（ｐＴＰＯ４７）亚克隆到Ｍ１３载体，测定全部ＤＮＡ顺序．它的碱基顺序与已报道的顺序完全相同，有一个１０５９ｂＰ的开放阅读框架可编码３５３个氨基酸．ｐＴＰＯ４７亚克隆到哺乳动物表达载体ｐＣＥＰ４，在哺乳动物２９３细胞系瞬时表达后，进行Ｎｏｒｔｈｅｒｎ杂交，结果显示有约１．４ｋｂ的转录产物．瞬时表达培养液腹腔注射小鼠后，可提高血小板计数４２．３％．血小板生成因子的克隆对进一步研究血小板生成调节以及该因子作用的分子机理具有重要意义．

A:thrombopoietin(TPO) cDNA encoding region of 1. 1kb was amplified from human fetal liver by RT-PCR and cloned. One of the clones,pTPO47, was subcloned into M13 vector and its complete sequence which includes a 1059 bp open-reading frame was determined. Then pTPO47 was subcloned into a mammalian expression vector, p(tEP4. A transcript of 1. 4 kb was identified by Northern blot analysis in the cell line 293 transfected with recombinant pCEP4. After daily intraperitoneal injection of the conditioned medium containing TPO produced by pCEP4 transfected cells into mice,the production of platelet increased 42. 3% more than that of the control,which were infected with the medium free of TPO. Futher research on regulaton of platlet production and its molecular mechanism will be greatly benefitted from the present study.