稳定干扰IFITM1表达的细胞系建立及对PRV、PCV2、TGEV在细胞内复制的影响

Establishment of interferon membrane protein IFITM1 knockdown cells and investigation of its effect on the replication of PRV,PCV2 and TGEV

为构建稳定干扰(IFITM1)基因表达的猪空肠上皮细胞系J2-KD(knockdown,KD),并研究干扰IFITM1表达后对伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪传染性胃肠炎病毒(TGEV)感染的影响。应用基因克隆技术,构建pLKO.1-EGFP-Puro-IFITM1重组载体,与慢病毒包装质粒共转染293FT细胞,产生绿色荧光蛋白标记的表达IFITMshRNA的慢病毒,48 h后收集病毒上清。用收获的慢病毒感染猪小肠上皮细胞(J2细胞)后,经过嘌呤霉素筛选及细胞有限稀释法,通过RT-PCR鉴定后,获得稳定干扰IFITM1基因的细胞系,并由MTT法验证干扰IFITM1表达对J2细胞生长无影响,将成功构建的干扰IFITM1表达的J2稳定细胞系命名为J2-KD。分别用PCV2、PRV和TGEV感染J2-KD细胞,用实时荧光定量PCR方法检测病毒的复制情况。结果显示,成功建立了稳定干扰IFITM1表达的J2细胞系(J2-KD);在J2-KD细胞中PCV2和TGEV病毒复制拷贝数出现升高,而PRV出现降低的现象。表明,干扰IFITM1基因明显抑制PRV病毒复制,而促进TGEV和PCV2的复制,这为进一步研究猪源IFITM1蛋白功能及阐明其抗病毒分子机制奠定了基础。

To establish a J2-KD(knockdown)cell line stably expressing interfered IFITM1 and study the effect of interference with IFITM1 gene on the infection of PCV2,PRV and TGEV.Gene cloning techniques were used to constructed pLKO.1-EGFP-Puro-IFITM1 recombinant vector,which was co-transfected into 293FT cells with lentiviral packaging plasmids psPAX2 and pMD2.G to produce green fluorescent protein labeled lentiviruses expression IFITM1shRNA,the viral supernatant was collected at 48 hours after post transfection.J2 cells were infected with the harvested lentiviruses,screened by puromycin and cloned via cell limited dilution.Real-time PCR identify that the cell lines with stable interference with IFITM1 gene were obtained,and via MTT method verify that interference with IFITM1 expression had no effect on the growth of J2 cells,the successfully constructed J2 stable cell line interfere with IFITM1 expression was named as J2-KD.PRV,PCV2 and TGEV infected J2-KD cells,respectively.Using real-time fluorescence quantitative PCR detect virus replication.The results showed that J2-KD cell line was successfully generated with interfered IFITM1 expression;the copy number of PCV2 and TGEV were increased,while PRV was decreased in J2-KD cell.Indicating that the interference of IFITM1 gene expression markedly inhibited the replication of PRV while promoted that of TGEV and PCV2,providing a basis for further study on the function of porcine IFITM1 protein and elucidates its antiviral mechanism.