小鼠红细胞分化相关因子cDNA片段的克隆与分析

Subtractive cDNA Cloning and Analysis of Murine Erythroid Terminal Differentiation Related Factor

目的克隆与小鼠红细胞终末分化相关因子的基因，并对其表达特性进行分析。方法用致贫血Ｆｒｉｅｎｄ病毒（ＦＶＡ）诱导ＢＡＬＢ／ｃ小鼠脾脏产生大量ＦＶＡ原红细胞，后者经分离纯化并在促红细胞生成素作用下进行体外培养。采用减除杂交与ＰＣＲ相结合的方法，用未经培养的ＦＶＡ诱导的原红细胞（０ｈＦＶＡ原红细胞）的ｃＤＮＡ对培养３６ｈ的ＦＶＡ诱导的成红细胞（３６ｈＦＶＡ成红细胞）的ｃＤＮＡ进行多次减除杂交和ＰＣＲ扩增，构建上述３６ｈｃＤＮＡ的质粒减除文库并进行差异筛选。对阳性克隆进行核苷酸序列测定及Ｎｏｒｔｈｅｒｎ印迹分析。结果获得１个４７２ｂｐ的ｃＤＮＡ片段，其中含有１个３０９ｂｐ的开放阅读框架，编码１０２个氨基酸。经ＧｅｎＢａｎｋ进行同源比较，未发现同源序列，已被ＧｅｎＢａｎｋ接受，（编号ＡＡ１１４３６９）。Ｎｏｒｔｈｅｒｎ印迹分析表明，该ｃＤＮＡ在培养３６ｈ的ＦＶＡ中、晚幼红细胞中呈差异表达，其ｍＲＮＡ全长约为５００～６００ｂｐ左右。将其暂命名为小鼠红细胞分化相关因子（ＭＥＤＲＦ）基因。结论ＭＥＤＲＦ基因在红细胞分化的中、晚幼阶段特异表达，表明ＭＥＤＲＦ是一个新的与红细胞终末分化相关的因子。

Objective Cloning and identification of cDNA related to erythroid terminal differentiation factor(MEDRF). Methods cDNA related to erythroid terminal differentiation from the Friend virus anemia (FVA) infected splenic erythroblasts of BALB/c mice were performed by using subtractive hybridization combined with PCR technique. The splenic proerythroblasts isolated were cultured in the presence of erythropoietin for 36 hrs. Subtractive cDNA clones of differentially expressed in the 36 hrs erythroblasts (sub cDNA 36) but absent in the uncultured proerythroblasts were observed. The sub cDNA 36 was then used for construction of subtractive cDNA library using the Bluescript SK(+) phage vector system, and differentially screened by 32 P labelled PCR generated probes. The positive clones were analyzed by nucleotide sequencing. Results The results indicated that a 472 bp cDNA fragment which contained a 309 bp reading frame from 51 to 359 coding 102 amino acids was identified and it has been accepted by GenBank as a new cDNA sequence that without comparable homology of existing sequences (accession number:AA114369). Northern blot analysis demonstrated that it was differentially expressed in the 36 hrs cultured intermediate late stages of erythroblasts. Conclusions The newly found cDNA, which expresses specifically in intermediate late stages of erythroblasts, not in stages of proerythroblasts, may be a new gene related to murine erythroid terminal differentiation.