MLKL基因敲除对猪伪狂犬病病毒复制的影响

doi:10.16431/j.cnki.1671-7236.2022.05.034

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (5): 1934-1941.doi: 10.16431/j.cnki.1671-7236.2022.05.034

MLKL基因敲除对猪伪狂犬病病毒复制的影响

谢思豪^1,2, 勾红潮², 卞志标², 李斌³, 蔡汝健², 臧莹安¹, 李春玲²

1. 仲恺农业工程学院, 广州 510225;
2. 广东省农业科学院动物卫生研究所, 广东省畜禽疫病防治研究重点实验室, 农业农村部兽用药物与诊断技术广东科学观测实验站, 广州 510640;
3. 广州市从化区动物卫生监督所, 广州 510900

收稿日期:2021-10-27 出版日期:2022-05-05 发布日期:2022-04-29
通讯作者: 臧莹安, 李春玲 E-mail:1134580900@qq.com;707989782@qq.com
作者简介:谢思豪,E-mail:3535297395@qq.com;勾红潮,E-mail:510089285@qq.com。
基金资助:
国家“十三五”重点研发专项课题（2018YFD0500804、2016YFD0500709）；国家自然科学基金青年科学基金项目（31902273）；广东省自然科学基金项目（2019A1515010757、2017A030310074）；广州市科技计划项目一般项目（201804010071）；科技创新战略专项（高水平农科院建设）（R2017YJ-YB2005、R2018QD-094）

Effects of MLKL Gene Knockout on Replication of Pseudorabies Virus

XIE Sihao^1,2, GOU Hongchao², BIAN Zhibiao², LI Bin³, CAI Rujian², ZANG Yingan¹, LI Chunling²

1. Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China;
2. Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
3. Guangzhou Conghua Animal Health Inspection Institute, Guangzhou 510900, China

Received:2021-10-27 Online:2022-05-05 Published:2022-04-29

摘要/Abstract

摘要： 【目的】构建混合谱系激酶结构域样(mixed lineage kinase domain-like，MLKL)基因敲除的PK-15细胞株(PK-15 MLKL-KO)，研究敲除MLKL基因对猪伪狂犬病病毒(Pseudorabies virus，PRV)复制的影响。【方法】根据MLKL序列设计特异性编辑位点，利用CRISPR/Cas9基因编辑技术构建MLKL-sgRNA编辑载体，转染至PK-15细胞，经嘌呤霉素药物筛选获得多克隆细胞系，通过有限稀释法获得PK-15 MLKL-KO单克隆细胞株。通过靶基因组PCR、测序和Western blotting验证MLKL基因在PK-15细胞上的敲除水平;采用Reed-Muench法检测病毒增殖水平;采用PI染色和荧光显微镜观察细胞坏死情况。【结果】试验成功构建MLKL-sgRNA载体，筛选出1株MLKL基因缺失647 bp的PK-15细胞株，Western blotting未检测到MLKL蛋白的表达。与PK-15细胞相比，PK-15 MLKL-KO细胞极显著或显著提高了PRV GD-WH(感染后36 h除外)和PRV Bartha-K61的病毒滴度(P<0.01;P<0.05)。PRV GD-WH和PRV Bartha-K61感染PK-15 MLKL-KO细胞后，坏死细胞明显减少。【结论】本研究构建了MLKL基因敲除的PK-15细胞株，与PK-15细胞相比，PK-15 MLKL-KO细胞显著提高了PRV GD-WH和PRV Bartha-K61的复制和存活能力，为PRV Bartha-K61疫苗生产过程中提高病毒产量提供了一种可行性策略。

关键词: MLKL基因; CRISPR/Cas9基因编辑技术; 敲除; PK-15细胞; 猪伪狂犬病病毒(PRV)

Abstract: 【Objective】 This study was aimed to construct knockout mixed lineage kinase domain-like (MLKL) PK-15 cell line,and investigate the effects of MLKL gene knockout on the replication of Pseudorabies virus (PRV).【Method】 The MLKL-sgRNA editing vector was constructed by designing specific editing sites based on MLKL sequences using CRISPR/Cas9 gene editing technology,and transfected into PK-15 cell.The polyclonal cell line was obtained by puromycin screening,and PK-15 MLKL-KO monoclonal cell line was obtained by limited dilution method.The knockout level of MLKL gene on PK-15 cell was verified by target genomic PCR,sequencing and Western blotting.Viral proliferation levels were detected using Reed-Muench method.PI staining and fluorescence microscopy were used to observe cell necrosis.【Result】 The results showed that one PK-15 cell line with a 647 bp deletion of MLKL gene was screened,and the expression of MLKL protein was not detected by Western blotting.Compared with PK-15 cell,PK-15 MLKL-KO cell extremely significantly or significantly increased the viral titers of PRV GD-WH (expect for 36 h post infection) and PRV Bartha-K61 (P<0.01 or P<0.05).After PRV GD-WH and PRV Bartha-K61 were infected with PK-15 MLKL-KO cells,the necrotic cells were significantly reduced.【Conclusion】 The MLKL gene knockout PK-15 cell line was constructed in this study.Compared with PK-15 cell,PK-15 MLKL-KO cell significantly improved the replication and survival of PRV GD-WH and PRV Bartha-K61,providing a feasible strategy to increase virus yield during PRV Bartha-K61 vaccine production.

Key words: MLKL gene; CRISPR/Cas9 gene editing technology; knockout; PK-15 cell; Pseudorabies virus (PRV)

中图分类号:

S852.65⁺9.1

谢思豪, 勾红潮, 卞志标, 李斌, 蔡汝健, 臧莹安, 李春玲. MLKL基因敲除对猪伪狂犬病病毒复制的影响[J]. 中国畜牧兽医, 2022, 49(5): 1934-1941.

XIE Sihao, GOU Hongchao, BIAN Zhibiao, LI Bin, CAI Rujian, ZANG Yingan, LI Chunling. Effects of MLKL Gene Knockout on Replication of Pseudorabies Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1934-1941.

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MLKL基因敲除对猪伪狂犬病病毒复制的影响

Effects of MLKL Gene Knockout on Replication of Pseudorabies Virus

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