LncRNA TCONS00179042对Marc-145细胞中PRRSV复制的影响

Effect of LncRNA TCONS00179042 on the replication of porcine reproductive and respiratory syndrome virus in Marc-145 cells

应用转染Lnc RNA TCONS_00179042过表达质粒pc DNA3.1-TCONS_00179042的Marc-145细胞,观察该Lnc RNA对PRRSV GSWW15毒株复制的影响。利用RT-PCR方法扩增出猪肺泡巨噬细胞（PAMs）被PRRSV GSWW15株感染后下调表达的Lnc RNA TCONS_00179042全长序列;通过q PCR、TCID50测定和Westernblot分析该Lnc RNA对PRRSV GSWW15株复制的影响;通过核质分离试验对PAMs细胞中Lnc RNA TCONS_00179042进行定位和半定量分析。RT-PCR检测结果显示,Lnc RNA TCONS_00179042长度约为430 bp。pc DNA3.1-TCONS_00179042过表达载体转染Marc-145细胞后,q PCR结果显示：该Lnc RNA的相对表达量约是对照组的3×105倍,表明Lnc RNA TCONS_00179042能在Marc-145细胞中过表达。利用PRRSV GSWW15株感染过表达Lnc RNA TCONS_00179042的Marc-145细胞后,q PCR结果显示：过表达Lnc RNA组PRRSV RNA拷贝数约为对照组的60%;TCID50检测结果显示：过表达Lnc RNA组PRRSV病毒滴度显著低于对照组,约为对照组的20%;Western-blot试验结果显示：过表达Lnc RNA组PRRSV N蛋白的表达量显著低于对照组。三组数据均显示,Lnc RNA TCONS_00179042能在Marc-145细胞中抑制PRRSV GSWW15株的复制。核质分离试验结果显示,不论是细胞质还是细胞核均有Lnc RNA TCONS_00179042存在,且细胞核中的表达量大于细胞质中。本研究结果表明,该Lnc RNA能在Marc-145细胞中抑制PRRSV的复制,能作为一种抗病毒分子参与调控宿主的抗病毒反应。

Marc-145 cells ectopically expressing LncRNA TCONS_00179042 were infected with porcine reproductive and respiratory syndrome virus（PRRSV） GSWW15 strain in order to observe the effect of LncRNA TCONS_00179042 on PRRSV GSWW15 replication.The full-length LncRNA TCONS_00179042 was amplified by RT-PCR.The effect of overexpression of L nc RNA TCONS 00179042 on replication of PRRSV GSWW15 strain inthe Marc-145 cells was detected by q PCR,TCID50 and Western-blot.The distribution of LncRNA TCONS_00179042 in porcine alveolar macrophages was analyzed by nuclear fraction isolation experiments.The q PCR results demonstrated that the relative expression level of LncRNA TCONS_00179042 in the pc DNA3.1-TCONS_00179042 transfected Marc-145 cells was about 3×105 folds of the control group,indicating that the LncRNA TCONS_00179042 was overexpressed in the Marc-145 cells.The q PCR results also showed that the PRRSV RNA copy number of the overexpressing LncRNA group was about 60% of that of the control group after infection with the PRRSV GSWW15 strain. In consistent with virus RNA level,the titer of PRRSV in TCONS_00179042 over-expression cells was significantly lower than that of the control cells.The result of Western-blot showed that the expression level of PRRSV N protein in LncRNA over-expression group was significantly lower than that in the control group.These results suggest that L nc RNA TCONS_00179042 inhibits the replication of PRRSV GSWW15 strain in Marc-145 cells.The nuclear separation experiment showed that the LncRNA TCONS_00179042 was located both in the cytoplasm and the nucleus,and the expression level in the nucleus was higher than that in the cytoplasm.The overexpression experiments demonstrated that LncRNA TCONS_00179042 has an inhibitory role in regula ting the replication of PRRSV GSWW15 strain in Marc-145 cells.