慢病毒介导稳定表达抑制PRRSV复制的shRNA细胞系的建立

doi:10.11843/j.issn.0366-6964.2016.11.016

Abstract

Abstract:

In the present study, Marc-145cell 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 PowerTM Packaging 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 (3.3 μg•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/Nsp9-6 and 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/Nsp9-6 specifically 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/NSP9-4 and Marc/pU6/NSP9-6, and the difference was obvious. These results demonstrate that Marc/pU6/NSP9-4 and Marc/pU6/NSP9-6 cell 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.

CLC Number:

S852.659.6

WU Jin-yan, TIAN Hong, SHANG You-jun, CHEN Yan, WANG Guang-xiang, LIU Xiang-tao, ZHANG Zhi-dong. Establishment of Cell Lines with Stable Expression of shRNA to Inhibit Porcine Reproductive and Respiratory Syndrome Virus Proliferation Using the Lentiviral Expression System[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(11): 2280-2287.

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

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