小鼠TLR4基因的克隆和序列分析

Cloning and sequence analysis of murine TLR4 gene

目的:克隆小鼠TLR4基因的全长编码区cDNA并进行序列分析。方法:利用RT-PCR方法,从Balb/c小鼠脾组织细胞mRNA中扩增TLR4基因全长cDNA,并将其连接到pcDNA3.1(+)载体上,进行测序和核酸分析。结果:扩增得到的小鼠TLR4基因cDNA全长2 508 bp,编码536个氨基酸,Blast结果分析表明,该cDNA与基因库中发表的序列(NM021297)具有100%的同源性。结论:获得小鼠TLR4基因的克隆,为进一步研究转基因免疫干预奠定了基础。

Objective： To clone all-length coding cDNA of murine. Methods： Total RNA as well as mRNA were isolated from splenic cells of Balb/e mice and the entire coding eDNA sequence of TLR4 was amplified by reverse transcription polymerase chain reaction （RT-PCR）. The TLR4 cDNA were cloned in to the peDNA3.1 （ ＋ ） vector. The identification was made by means of restriction enzym eanalysis, PCR and DNA sequencing. Results： The murine entire coding cDNA of 2 508 bp was enamplified from spleen cells. There was 100% homogenous nueleotide sequence as compared with standard sequence（NM021297） of TLR4 from Genbank. Conclusion： We successfully cloned the cDNA of murine TLR4. It will be the basis for studying immune interference of transferring gene.