鸡源AID基因的克隆及其抗新城疫病毒作用

Identification of chicken activation-induced cytidine deaminase (aid) and its anti-viral effect to Newcastle disease virus

APOBEC家族成员中AID蛋白具有DNA/RNA编辑酶功能,同时具有抑制病毒增殖的作用,但具体机制尚不明了。本研究首先根据NCBI公布的鸡全基因组序列进行生物信息学分析,预测鸡AID的基因序列并通过设计PCR引物,成功在鸡法氏囊cDNA样品中扩增出鸡AID基因(chAID)。通过建立并优化chAID的实时定量RT-PCR(rRT-PCR)检测方法,对鸡体内AID基因的表达以及其分布情况进行检测;同时通过Western blot试验对chAID蛋白在鸡细胞中的表达进行检测。试验显示,chAID基因能够在鸡细胞以及组织脏器中正常表达。新城疫病毒(NDV)感染鸡原代成纤维细胞、淋巴细胞后能够引起内源性chAID基因水平的上调;SPF鸡感染NDV后,chAID基因在不同组织脏器中均出现上调现象,以免疫器官最为显著。为了进一步验证chAID基因对NDV增殖的影响,进一步检测在细胞中过表达chAID基因对NDV增殖的影响。研究结果表明:重组chAID蛋白具有抗NDV病毒活性,本研究为进一步研究鸡chAID奠定了基础。

Activation-induced cytidine deaminase(AID),belonging to the APOBEC family,as the function of RNA editing and inhibiting virus propagation.The mechanism of AID function is not clear yet,bioinformatics analysis based on the chicken whole genome to predict the chicken AID(chAID)gene sequence were carried out in this study.The predicted chAID gene was successfully isolate from cDNA of chicken fabricius’bursa.The real-time RT-PCR method specific for chAID was established.The expression of chAID protein in chicken cells and organs was determined by Real-time PCR and Western blot assay.In NDV-infected chicken fibroblasts and lymphocytes,chAID gene was highly expressed.The same high expression of chAID was observed in organs of chicken infected with NDV.Furthermore,the results showed that in cell over-expressing chAID,the replication of NDV were inhibited.The detailed mechanism of chAID mediated anti-NDV effect need to be further studied.