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

摘要/Abstract

摘要：

利用Gateway技术和慢病毒表达系统将山羊淋巴细胞信号活化因子SLAM稳定整合在Vero细胞基因组染色体上，建立稳定表达可增强小反刍兽疫病毒（PPRV）复制的SLAM的Vero阳性细胞亚克隆，并验证阳性细胞亚克隆Vero/SLAM表达SLAM的活性、遗传稳定性及其对PPRV复制的增殖效果。分离山羊外周血淋巴细胞，提取基因组Total RNA，一步RT-PCR获得完整ORF的SLAM基因，采用BP及LR位点的基因重组技术，构建入门载体pDONR/SLAM并获得表达骨架pDEST/SLAM，与Packaging Mix pLP1、pLP2及VSV-G共转染293-FT细胞，获得SLAM复制缺陷型慢病毒样粒子，用其感染Vero细胞，杀稻瘟菌素抗性筛选获得阳性细胞克隆，通过靶标基因扩增、间接免疫荧光、激光扫描共聚焦显微技术、Western blot免疫印迹检测技术、致细胞病变效应及Real-time RT-PCR等技术分别验证SLAM基因组整合、转录，SLAM蛋白表达、反应活性以及PPRV在表达SLAM阳性细胞亚克隆上的增殖效果。结果显示：SLAM受体基因被稳定整合在Vero细胞基因组染色体上，该受体蛋白表达于细胞周质，建立的细胞连续传代不丢失，接种病毒后致细胞病变时间由4~7 d缩短为3.5 d，TCID₅₀·0.1 mL^-1由4.25增加为5.67，相对于正常Vero细胞，整合有SLAM的Vero细胞，由于表达了PPRV特异的细胞受体使PPRV对其易感性显著增强。成功建立一株稳定表达靶向增强PPRV复制的Vero/SLAM细胞：该细胞表达的SLAM具有明显增强PPRV复制的功能，不仅可以从细胞模型水平阐明增强PPRV复制的关键靶基因，也为PPRV感染引起宿主细胞的变化以及对机体的致病机制等提供研究工具。

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.

中图分类号:

S855.3
Q78

吴锦艳, 田宏, 蒙学莲, 惠小婷, 王耀杰, 关玉华, 尚佑军, 刘永生, 张志东. 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[J]. 畜牧兽医学报, 2019, 50(1): 218-226.

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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