东方田鼠三个脂肪肝相关基因的克隆及分析

Acquisition and Analysis of Three cDNA Full-Length Sequences from the Liver of Microtus fortis

目的克隆CYP2E1、CYP2D5、ECHS1三个与非酒精性脂肪肝可能相关基因的cDNA全长基因,为进一步研究非酒精性脂肪肝的发病机制提供依据。方法利用SMART技术构建东方田鼠肝脏cDNA质粒文库,通过PCR方法筛选文库,获得目的菌落,用pBluescript Ⅱ SK通用引物M13R测序,获得基因全长序列,并进行序列分析比较。结果筛选了CYP2E1、CYP2D5、ECHS1三个基因,并获得了它们的全长cDNA序列:CYP2E1基因cDNA全长为1685bp,有1482 bp的完整开放阅读框,编码494个氨基酸;CYP2D5基因cDNA全长为1690 bp,有1514 bp的完整开放阅读框,编码504个氨基酸;ECHS1基因cDNA全长为1013 bp,有873 bp的完整开放阅读框,编码290个氨基酸。这三个基因的核酸序列及其推测的氨基酸序列与人、大小鼠序列高度同源。结论获得了东方田鼠CYP2E1、CYP2D5、ECHS1三个基因全长cDNA序列,并已在Genbank中登录(GQ507485、GQ507486、GQ845171),为东方田鼠非酒精性脂肪肝模型的研究奠定了基础。

Objective To obtain the full-length cDNA sequences of CYP2E1,CYP2D5,ECHS1,which may be related with non-alcoholic fatty liver disease,from Microtus fortis.Methods To construct Microtus fortis liver cDNA plasmid library using SMART technique,to get the purposed colonies through screening libraries by PCR,and to obtain their full-length cDNA sequences by sequencing with pBluescript II SK universal primers M13R.Results Three full-length cDNA sequences of Microtus fortis,CYP2E1,CYP2D5 and ECHS1 were obtained.The CYP2E1 cDNA was 1685 bp in length and contained a 1482 bp open reading frame（ORF） encoding a 494 amino acids.The CYP2D5 cDNA was 1690 bp in length,and contained a 1514 bp ORF encoding 504 amino acids.The ECHS1 cDNA was 1013 bp in length,and containsed an 873 bp ORF encoding 290 amino acids.Sequence analysis revealed that the identity of the three cDNA sequences and deduced amino acids among Microtus fortis,Homo sapiens,Mus musculus and Rattus norvegicus was high.Conclusion The full-length cDNA sequences of CYP2E1,CYP2D5,ECHS1 were obtained from Microtus fortis liver cDNA library,and the gene sequences have been deposited in GenBank（GQ507485,GQ507486,GQ845171）,which may lay the foundation for researchies of pathogenesis of non-alcoholic fatty liver disease in Microtus fortis models.