慢病毒介导稳定表达增强小反刍兽疫病毒复制的山羊Vero/SLAM细胞系的建立

doi:10.11843/j.issn.0366-6964.2019.01.025

Abstract

Abstract:

By the Gateway technology and the lentiviral expression system, goat signaling lymphocyte activation molecule (SLAM,also known as CD150) was stably integrated into the genome chromosome of Vero cell,to establish a Vero/g-SLAM positive cell clones, with which the ability of replication of peste des petits ruminants virus (PPRV) can be improved. On the other hand, to verify the activity, genetic stability and proliferation of PPRV expressing SLAM of Vero/g-SLAM. Peripheral blood lymphocytes were isolated from goat, of which genomic total RNA was extracted, SLAM gene with complete ORF were obtained by one step RT-PCR. Using of the BP and LR loci gene recombination technology to construct the entry clone vector pDONR/SLAM and expression skeleton of pDEST/SLAM, with packaging plasmids pLP1, pLP2 and VSV-G to co-transfect 293-FT cells to produce a lentiviral stock, use the lentiviral stock to infect Vero cell. The Vero cells with SLAM were named Vero/SLAM, and were resistant to the blasticidin (3.5 μg·mL^-1). By the target gene amplification, Confocal laser scanning microscope, indirect immunofluorescence, Western blot and cytopathic effect, the SLAM genome integration, transcription, expression and reaction activity of SLAM protein, and PPRV proliferation effect in Vero/SLAM were verified respectively. Results were as follows:SLAM was stably integrated in the genome of Vero cells; The receptor protein was expressed in the periplasmic part of the cell; After continuous subpassages of the cells, SLAM are not lost; Vero/SLAM were inoculated virus The cytopathic time was shortened from 4-7 d to 3.5 d, and TCID₅₀·0.1 mL^-1 increased from 4.25 to 5.67. Compared with the normal Vero cells, Vero cells that integrate SLAM have significantly enhanced their susceptibility to PPRV because of expression of PPRV specific cell receptors. In this study, a new cell line, Vero/SLAM cells was established successfully. The SLAM expressed by this cell has the obvious function of enhancing PPRV replication, the critical target genes that enhance PPRV replication can be elucidated at the cellular model level. It also provides information for the study of host cell changes caused by PPRV infection and the pathogenesis of the organism.

Key words: peste des petits ruminants virus, SLAM, Vero cells, Gateway technology, lentiviral expression system

CLC Number:

S855.3
Q78

WU Jinyan, TIAN Hong, MENG Xuelian, HUI Xiaoting, WANG Yaojie, GUAN Yuhua, SHANG Youjun, LIU Yongsheng, ZHANG Zhidong. Establishment of Vero/SLAM Cell Lines with Stable Expression SLAM to Enhanced PPRV Replication Using the Lentiviral Expression System[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 218-226.

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Establishment of Vero/SLAM Cell Lines with Stable Expression SLAM to Enhanced PPRV Replication Using the Lentiviral Expression System

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