小鼠白血病抑制因子真核表达载体的构建及测序分析

Construction of Eukaryotic Expression Vector of Mouse LIF cDNA and Sequence Analysis of Mouse LIF

运用RT-PCR技术,克隆含信号肽和不含信号肽的小鼠分泌型白血病抑制因子cDNA,通过pMD18-T simple载体和pBS-T载体过渡,分别构建了真核表达载体pSecTag-mlif(sp+)和pSecTag-mlif(sp-),酶切进行初步鉴定。利用Blast程序,搜索NCBI GeneBank中与构建表达载体中编码MLIF cDNA的同源序列,除在编码区216bp处碱基为G和在318bp处由G突变为T外,编码LIF基因的其余序列与已发表的完全一致。运用DNAMAN软件对翻译水平进行预测,结果发现这一突变位点并不影响蛋白的翻译。

Mouse secreted LIF cDNA with and without signal peptide were cloned from mouse livers by RT-PCR, and then subcloned into pBS-T vector and pMD18-T simple vector. Digesting fragments were recovered and inserted into pSecTag/Hygro with molecular cloning technique. Restrictive enzymes digestion analysis and DNA sequence results revealed that the LIF gene was cloned into eukaryotic expression vector pSecTag/Hygro successfully. The expression vectors which were constructed were called pSecTagmlif（sp＋） and pSecTag-mlif（sp ）. The sequence encoding mouse LIF gene which were cloned is consistent with published sequence in GeneBank except that the encoding sequence had two mutations in 216bp and in 318bp. Using DNAMAN software, we found that the two mutations did not affect protein translation.