摘要
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.
出处
《世界华人消化杂志》
CAS
北大核心
2007年第21期2284-2289,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金
No.30600278
关键词
RELMβ基因
组织特异性
结肠
基因表达
免疫印迹
逆转录聚合酶链反应
Resistin-like molecule beta
Tissue specificity
Colon
Gene Expression
Western blotting
Northern blotting
Reverse transcriptase-polymerase chain reaction