IRF8新发移码突变的基因功能分析  

Functional characterization of de novo frameshift mutation in IRF8

作　　者：李乐盈 陈尧 周维涛 何晨 张端午 钱莉玲 LI Le-ying;CHEN Yao;ZHOU Wei-tao;HE Chen;ZHANG Duan-wu;QIAN Li-ling(Department of Respiratory Medicine,Children's Hospital/National Children's Medical Center,Shanghai 201102,China;Institute of Pediatrics,Children's Hospital/National Children's Medical Center,Shanghai 201102,China;Institutes of Biomedical Sciences,Fudan University,Shanghai 200032,China)

机构地区：[1]国家儿童医学中心/复旦大学附属儿科医院呼吸科,上海201102 [2]国家儿童医学中心/复旦大学附属儿科医院儿科研究所,上海201102 [3]复旦大学生物医学研究院,上海200032

出　　处：《复旦学报（医学版）》2024年第3期359-367,共9页Fudan University Journal of Medical Sciences

基　　金：上海市卫健委青年项目(20204Y0101)。

摘　　要：目的对反复肺炎患儿队列病因筛查中发现的干扰素调节因子8基因(interferon regulator factor 8,IRF8)新发突变(c.1266dupA)进行分子细胞水平的功能研究与验证。方法针对IRF8突变序列构建野生与突变蛋白过表达载体,通过质粒转染或构建慢病毒感染不同工具细胞,通过qPCR、Western blot、免疫荧光等实验手段检测其表达差异及对下游炎症相关基因表达调控的改变。结果IRF8野生及突变过表达载体构建成功,且转染效率可观,包装成的慢病毒感染效率亦较高。突变基因IRF8(c.1266dupA)相较于野生型IRF8转录水平降低,表达蛋白的相对分子质量略增大,表达量降低,突变蛋白IRF8mut相对野生型IRF8蛋白核质比增加,IRF8(c.1266dupA)对下游ISRE元件的抑制功能增强。结论IRF8新发移码突变属于功能获得型(gain of function,GOF)突变。Objective To study and verify the function of de novo interferon regulatory factor(IRF8)frameshift mutation detected in an etiology screening of the cohort of children with recurrent pneumonia at the molecular level.Methods The recombinant overexpression plasmids with wildtype or mutated IRF8 genes were constructed to transiently transfect HEK293T cells,or packed into lentivirus to infect two kinds of immune cell lines.Q-PCR,Western blot,immunofluorescence and other experimental assays were performed to explore the differences of expression and the regulatory effect on downstream genes associated with inflammation.Results The recombinant vectors with wildtype or mutated IRF8 genes were constructed successfully,and the efficiency of transfection by plasmids and infection by packed lentivirus was remarkable as well.Compared with wildtype,the molecular weight of IRF8 variant was slightly increased,while the expression level presents in opposition,even if on transcription level.Moreover,the localization of IRF8 variant was detected in abundance in nucleus rather than cytoplasm,and its inhibition effect was enhanced on the downstream ISRE element in comparison with the wildtype IRF8 protein.Conclusion The de novo frameshift mutation was presumed as gain-of-function(GOF)mutation.

关 键 词：干扰素调节因子8(IRF8) 移码突变 反复感染 转录调控 

分 类 号：R725[医药卫生—儿科]