肺组织特异性基因HIMF的克隆及其真核表达载体的构建

Cloning of Lung-specific Gene Hypoxia-induced Mitogenic Factor and Construction of Its Eukaryotic Expression Vector

目的克隆小鼠肺组织特异性表达基因缺氧诱导丝裂原因子(hypoxia-induced mitogenic factor,HIMF),并构建其真核表达载体。方法采用Western blot法检测HIMF基因在小鼠不同脏器中的表达;从小鼠肺组织中提取总RNA,采用RT-PCR法扩增全长HIMF cDNA,克隆至T载体后进行核酸序列分析,并将其正向插入到真核表达载体pcDNA3.1/Zeo(+)的限制性内切酶位点EcoRⅠ;重组子在阳离子脂质体Lipofectamine 2000的介导下瞬时转染胚胎纤维母细胞NIH/3T3、肺Ⅱ型上皮细胞MLE-12、血管内皮细胞SVEC4-10,用Western blot及RT-PCR法检测细胞HIMF表达水平。结果HIMF特异性表达于小鼠肺组织,而其他脏器未见表达。成功克隆了小鼠HIMF全长cDNA(336bp),与GenBank报道的序列一致。真核表达载体pcDNA3.1-HIMF转染细胞后,细胞内HIMF mRNA和蛋白水平均显著增高(P<0.01)。结论从小鼠肺组织中克隆出HIMF这一组织特异性基因,为深入研究HIMF的生物学功能及其在肺部疾病靶向性生物治疗中的作用奠定了基础。

Objective To clone hypoxia-induced mitogenic factor （HIMF）, a lung tissue-specific gene, and construct its eukaryotic expression vector. Methods HIMF expression in organs and tissues of mice was detected by using Western blot. Total RNA was extracted from mouse lung tissues. Full length cDNA of HIMF gene was amplified by reverse transcription polymerase chain reaction （RT-PCR）, inserted into T/A vector, validated by nucleic acid sequencing, and subcloned into the EcoR Ⅰ restrictive site of eukaryotic vector pcDNA3.1/Zeo（＋）. Under the induction of Lipofectamine 2000, the recombinant was transferred into embryonic fibroblast cells NIH/3T3, alveolar type Ⅱ cells MLE-12 and vascular endothelial cells SVEC 4- 10. The expression levels of HIMF were detected by Western blot and RT-PCR. Results The HIMF protein was specifically expressed in mouse lung rather than other organs. The mouse full-length HIMF cDNA （336 bp） was successfully cloned, with the same sequence as ＂reported in GenBank. Transfection of cells with eukaryotic vector pcDNA3.1-HIMF resulted in enhanced HIMF mRNA and protein levels （P〈0.01）. Conclusion The tissue-specific gene HIMF was cloned from mouse lung tissues, which established a basis for further exploring the biological functions of HIMF and potential strategies for targeted biological therapy of lung diseases.