中文摘要：

目的探讨获取小鼠Lin28蛋白的方法。方法 提取8.5 d ICR小鼠胚胎mRNA后反转录为cDNA序列,用一对两端引入特定酶切位点（NcoⅠ及XhoⅠ）引物,从该cDNA中扩增出Lin28基因编码区序列;将获得的Lin28基因编码区序列克隆到pMD18-T载体上。对质粒双酶切回收其中Lin28基因片段,与pET-30a（＋）载体相连接并转化Rosetta（DE3）型大肠杆菌,用IPTG诱导表达,最后采用SDS-PAGE对表达结果进行分析。结果对所克隆的Lin28蛋白编码区的DNA序列分析表明,Lin28 CDS区包括终止密码子在内为630 bp,与参照DNA（NM_145833）相比同源性为99.37%,与参照氨基酸序列相比同源性为100%;在IPTG诱导下pET-30a（＋）-Lin28重组质粒可表达与预期相符的约为27.5×103的蛋白质。结论利用克隆的小鼠Lin28基因,采用原核表达方法,成功获得小鼠Lin28蛋白,为进一步开展以重组蛋白诱导体细胞重编程研究奠定基础。

英文摘要：

Objective Lin-28 is a gene recently shown to be involved in the conversion of somatic cells to induced pluripotent stem cells.The aim of this study was to clone Lin28 gene and analyzed its expression in E.coli so to make an effort on the further study of its function.Methods Lin28 gene was cloned from the cDNA amplified from 8.5-day mouse embryos mRNA,and ligated with linear vector pMD18-T.The recombinant plasmid was digested and the segment with Lin28 gene was purified then subcloned into prokaryotic expressing vector pET-30a（＋）.Results The recombinant plasmid was transfected into E.coli strain Rosetta（DE3） and its expression induced by IPTG was identified as well analyzed by SDS-PAGE.Compared with the DNA（NM-145833）,the homology was 99.37%,and compared with the amino acid sequence,the homology was 100%.Conclusions The coding region of Lin28 in a length of 630 bp is successfully harvested and recombinant protein with 27.5 kD just as anticipated.