稳定表达流感病毒PR8株NS1蛋白MDCK细胞系的建立

ESTABLISHMENT OF A STABLE MDCK CELL LINE EXPRESSING NS1 PROTEIN OF PR8 INFLUENZA VIRUS

根据流感病毒A/Puerto Rico/8/34株NS1基因的核苷酸序列设计引物,PCR扩增后,将NS1完整开放阅读框分别克隆于pMX载体和PET30a载体,成功构建了pMX-PR8-NS1和PET-PR8-NS1重组质粒。将PET-PR8-NS1重组质粒转化Jm109感受态大肠杆菌,诱导表达获得重组NS1蛋白免疫小鼠制备多抗血清。同时,将逆转录病毒载体系统pMX-PR8-NS1、pCI-NF-KB、PMDSV和MDSV共转染293T细胞,制备逆转录病毒样粒子。将逆转录病毒样粒子感染MDCK细胞,利用嘌呤霉素进行抗性筛选。然后经过PCR、RT-PCR、间接免疫荧光鉴定,获得稳定表达PR8病毒NS1蛋白的细胞系,将之命名为MDCK-PR8-NS1细胞系。该细胞系的建立有望为深入开展流感病毒NS蛋白生物学功能的研究以及为扩增NS1基因删除的流感病毒提供了有利的工具。

In order to amplify the entire open reading frame of NS1 gene,the specific primers were designed according to the nucleotide sequence of NS gene of A/ Puerto Rico/ 8/ 34 Influenza virus were designed.The PCR product of NS1 gene was inserted into pMX vector and PET30a vector,and recombinant plasmids pMX-PR8-NS1 and PET-PR8-NS1 were generated.After being expressed in E.coli,NS1 protein was purified and injected into BALB/c mice for preparation of NS1 specific polyclonal antibody.Meanwhile,to produce retrovirus-like particles,plasmids pMX-PR8-NS1,pCI-NF-KB,PMDSV and MDSV were co-transfected into 293T cells.Then the retrovirus-like particles which produced and released into culture supernatants from 293T cell transfected were used to infect MDCK cells.The cells inserted with expected exogenous gene were selected by puromycin.After analyzed by PCR（using cell genome as template）,RT-PCR（using total cell RNA as template） and immuno-fluorescence assay,a cell line（MDCK-PR8-NS1） was obtained with can express NS1 protein stably.This cell line may provide a useful tool to study the function of NS1 gene or protein.