鸡APOBEC4稳定表达细胞系的建立与应用

Establishment and Application of BHK21 Cell Line Stably Expressing Chicken APOBEC4

鸡APOBEC4是最新发现的载脂蛋白B mRNA编辑酶(APOBEC)家族成员,功能尚不明确。由于具有保守的胞苷脱氨酶基序,该家族蛋白能够诱导机体DNA或RNA发生广泛的胞嘧啶脱氨基突变,参与到机体免疫过程中去,在机体先天性免疫与适应性免疫中均发挥重要作用。这种RNA编辑作用亦可诱导病毒基因组突变,产生种类众多的突变株,但由于每种突变在群体中的比例极小,自然状态下会被优势准种掩盖难以检测,因此需要能够稳定表达该蛋白的细胞系作为模型进行研究。本实验室前期结果表明,鸡源APOBEC4主要在免疫细胞中表达,而传染性支气管炎病毒、流感病毒感染能够显著刺激鸡APOBEC4的大幅上调。为研究鸡APOBEC4编辑作用对病毒复制的影响,本研究利用慢病毒包装方法,将鸡源APOBEC4基因导入BHK21细胞,通过Real-time quantitative PCR和Western blot方法进行鉴定,得到能够稳定表达鸡APOBEC4的BHK21细胞系BHK21-APOBEC4,并发现鸡传染性支气管炎病毒(IBV)Beaudette株和禽流感病毒(AIV)H9N2株在该细胞系上的复制较对照组受到显著抑制,为进一步研究APOBEC4的生物学功能奠定基础。

Chicken APOBEC4 is the newly discovered member of the APOBEC(apolipoprotein B mRNA editing enzyme)protein family,whose function is unknown.This protein family plays an important role in both innate and adaptive immunity.Owing to the conserved zinc-coordinating deaminase domain that catalyzes the deamination of cytidine to uridine(C→U/G→A)in DNA/RNA,APOBEC proteins can induce a wide range of cytosine deamination mutations in vivo and participate in immune response.It has been found that some viral genomes can be edited by APOBECs as well,suggesting its involvement in virus evolution.The mutations on viral genome introduced by APOBECs are difficult to be detected due to their low rate in the population.Thus,the cell lines stably expressing APOBEC proteins are required for related researches.Our preliminary studies indicated that the distribution of chicken APOBEC4 was different in mammals and the overexpression of chicken APOBEC4 in cells showed obvious inhibitory effect on IBV and H9N2.In order to study the effect of chicken APOBEC4 editing on virus replication,its gene was transduced into BHK21 cell line through lentiviral packaging method.The BHK21 cells then stably expressed chicken APOBEC4 as tested by real-time quantitative PCR and Western blot.The results showed that the replication of IBV and H9N2 on this BHK21-APOBEC4 cell line was significantly inhibited as compared with the control group.Therefore,the present study laid a foundation for further study of the biological function of APOBEC4.