小鼠LRRFIP1基因的克隆及LRRFIP1天然免疫模式识别功能的研究

Cloning of mouse LRRFIP1 gene and study on functions of LRRFIP1 in innate immunity pattern recognition

应用RT-PCR技术,从小鼠脾淋巴细胞中成功克隆了LRRFIP1基因,并对其遗传演化关系及LRRFIP1的分子结构特征进行了分析。结果显示,克隆的基因开放阅读框全长2 190bp,编码729个氨基酸,与大鼠、猪、牛等哺乳动物LRRFIP1相应序列的同源性为90.5%~46.5%。将克隆的小鼠LRRFIP1基因亚克隆至真核表达载体pCMV-Tag 2B,转染HEK293T细胞后,双荧光素酶报告系统证实,在poly(dA-dT)和poly(dG-dC)刺激下,过表达小鼠LRRFIP1能够通过激活IRF3而显著增强IFN-β的表达。结果表明,小鼠LRRFIP1能够识别富含A/T或C/G的DNA序列,在宿主抗DNA病毒感染的天然免疫反应中可能发挥重要作用。

The coding sequence of mouse LRRFIP1 gene was amplified from splenic mononuclear cells by RT-PCR.In addition,the genetic evolution and molecular structure of the mouse LRRFIP1 gene were analyzed.In result,the ORF of this sequence is 2 190 bp in size,encoding 729 amino acids.The mouse LRRFIP1 gene exhibited identity with the corresponding sequences of rat,pig,cattle,ranging from 46.5% to90.5%.The cloned LRRFIP1 gene was subcloned into the eukaryotic expression vector pCMV-Tag 2B.The recombinant plasmid was then transfected into the HEK293 Tcells.The IRF3 and IFN-βluciferase reporter systems verified that overexpression of mouse LRRFIP1 significantly induced the activity of IFN-βby activating transcription factor IRF3 with the stimulation of poly(dA-dT)and poly(dG-dC).These results indicated that mouse LRRFIP1 was able to recognize A/T or G/C-rich DNA sequence,and may play a vital role in host innate immune responses against DNA virus infections.