慢病毒介导稳定表达抑制PRRSV复制的shRNA细胞系的建立

Establishment of Cell Lines with Stable Expression of shRNA to Inhibit Porcine Reproductive and Respiratory Syndrome Virus Proliferation Using the Lentiviral Expression System

利用慢病毒表达技术将稳定持续抑制PRRSV复制的shRNA导入Marc-145细胞,建立稳定表达靶向抑制PRRSV复制的shRNA的Marc-145阳性细胞克隆,并从细胞模型水平阐明抑制PRRSV复制的关键靶基因的干扰效果。采用LR重组技术,将pENTR/U6/Nsp9-4、pENTR/U6/Nsp9-6及pENTR/U6/-CON分别与pDEST载体进行LR重组,获得表达骨架,重组后的表达载体在转染试剂介导下与已经优化的辅助质粒Vira PowerTM Packaging Mix共转染293-FT包装细胞,获得慢病毒样粒子,并用其感染Marc-145细胞,杀稻瘟菌素抗性筛选获得阳性细胞克隆,通过PCR、CPE、TCID50、Real-time PCR和间接免疫荧光试验等方法分别验证上述细胞株的稳定整合以及其表达的shRNA对PRRSV增殖的抑制效果。结果显示:优势干扰序列和无关序列被稳定整合在靶细胞基因组上,无论观察细胞病变效应、免疫荧光产生情况、致细胞半数感染量还是实时定量分析相对表达量,与正常Marc-145和整合有无关序列的两株阴性对照细胞相比,整合有NSP9-4和NSP9-6的两株细胞由于表达了靶向抑制PRRSV复制的shRNA,PRRSV对其易感性降低,而且差别显著,分别将其命名为Marc/pU6/NSP9-4和Marc/pU6/NSP9-6。经验证,本研究成功构建两株稳定表达靶向抑制PRRSV复制的shRNA细胞——Marc/pU6/NSP9-4、Marc/pU6/NSP9-6,同时获得一株表达无意义shRNA的Marc/pU6-CON辅助细胞,该细胞表达的shRNA具有明显抑制PRRSV复制的功能,不仅可以从细胞模型水平阐明抑制PRRSV复制的关键靶基因,也为PRRSV感染引起宿主细胞的变化以及对机体的致病机制等研究提供资料。

In the present study,Marc-145 cell lines with stable expression of shRNA were established by lentiviral expression system to inhibit replication of porcine reproductive and respiratory syndrome virus（PRRSV）,and its interference suppression effect to PRRSV key target genes was clarified from the level of cell model.LR recombination reaction between pENTR/U6/Nsp9-4（pENTR/U6/Nsp9-6,or pENTR/U6/-CON,respectively）and pDEST were done by using the LR technology to obtain expression skeleton.Expression vector and auxiliary plasmid Vira Pow-er TMPackaging Mix were cotransfected into 293-FT cells by Lipofectamine 2000,respectively,to gain Lentiviral supernate and to infect Marc-145 cells.After 48 h,the cells were incubated in standard culture medium including blasticidin（3.3μg·mL-1）,colonies with blasticidin were obtained when cells were cultured in selection medium within 14 d,monoclonal were expanded,passaged,screened to get monoclonal cell lines.The integration of the obtained monoclonal cell lines were identified by PCR,CPE observation and TCID50 determination,and the inhibition effect of the cell lines with shRNA to PRRSV proliferation were detected by indirect immunofluorescence test and real-time PCR methods.pENTR/U6/Nsp9-4,pENTR/U6/Nsp9-6and Marc/pU6-CON had been integrated into the chromosome of Marc-145 cells and expressed stably at high level.Results showed that the shRNA expressed by two cells with pEN/U6/Nsp9-4,pEN/U6/Nsp9-6specifically suppressed PRRSV proliferation,respectively.The infection rates of PRRSV to the negative control and normal cells were higher than those to the recombinant Marc/pU6/NSP9-4and Marc/pU6/NSP9-6,and the difference was obvious.These results demonstrate that Marc/pU6/NSP9-4and Marc/pU6/NSP9-6cell lines can effectively express shRNA to inhibit the replication and proliferation of PRRSV.It helps to clarify the key target genes that suppress PRRSV replication,and is expected to construct transgenic animals based on the siRNA targeted PRRSV.