结肠特异性基因RELMβ的克隆及其真核表达载体构建

Cloning of colon-specific gene resistin-like molecule beta and construction of its eukaryotic expression vector

目的:观察克隆小鼠结肠特异性基因RELMβ,并构建其真核表达载体.方法:采用Western blot、Northern blot方法检测RELMβ基因在小鼠不同脏器中的表达;从小鼠结肠组织中提取总RNA,采用逆转录聚合酶链反应(RT-PCR)扩增RELMβ全长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EcoR I:重组子在阳离子脂质体Lipofectamine 2000的介导下瞬时转染小鼠胚胎纤维母细胞NIH/3T3、结肠癌细胞系CT26,Western blot及RT-PCR法检测细胞RELMβ表达水平.结果:RELMβmRNA和蛋白特异表达于小鼠结肠,而其他脏器未见表达.成功克隆了小鼠RELMβ全长cDNA(318 bp),与已报道序列的同源性高达99%,获得GenBank登录号DQ157777.真核表达载体pcDNA3.1-RELMβ转染细胞后,细胞内RELMβmRNA和蛋白水平均显著增高(P<0.01).结论:从小鼠结肠组织中克隆出RELMβ特异性表达基因,为深入研究RELMβ基因的生物学作用及其在结肠疾病靶向治疗中的作用奠定了基础.

AIM： To clone mouse resistin-like molecule beta （RELMβ）, a colon-specific gene, and construct its eukaryotic expression vector. METHODS： RELMβ expression in diverse organs and tissues in mice was investigated by Western and Northern blotting. Total RNA was extracted from mouse colon tissues. Full-length cDNA of the RELMβ gene was amplified by reverse transcriptase-polymerase chain reaction （RT-PCR）, inserted into T/A vector, validated by nucleic acid sequencing, and subcloned into the EcoRⅠ restriction site of eukaryotic vector pcDNA3.1/Zeo（＋）. Under induction with Lipofectamine 2000, the recombinant cDNA was transferred into mouse embryonic fibroblast NIH/3T3 cells and colon cancer CT26 cells. Cellular expression levels of RELMβ were measured with Western blotting and RT-PCR. RESULTS： RELMβ mRNA and protein were specifically expressed in mouse colon rather than in other organs. The mouse full-length RELMβ cDNA （318 bp） was successfully cloned, with 99% homology with the reported sequence. A GenBank access number was achieved as DQ157777. RELMβ mRNA and protein levels were enhanced after transfection of cells with eukaryotic vector pcDNA3.1-RELMβ （P 〈 0.01）. CONCLUSION： The colon-specific gene RELMβ was cloned from mouse colon tissues, which established a basis for further exploring the biological functions of RELMβ and potential strategies for targeted therapy of colon diseases.