猪丁型冠状病毒在悬浮培养猪肾细胞LLC-PK1上的增殖特性分析

doi:10.11843/j.issn.0366-6964.2022.06.035

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (6): 2024-2028.doi: 10.11843/j.issn.0366-6964.2022.06.035

猪丁型冠状病毒在悬浮培养猪肾细胞LLC-PK1上的增殖特性分析

李厚伟¹, 王蕾², 张先锋², 胡慧³, 张真真², 张云飞³, 刘林涛³, 姬星宇², 胡永浩^1*

1. 甘肃农业大学动物医学院，兰州 730070;
2. 商丘美兰生物工程有限公司，柘城 476200;
3. 河南农业大学动物医学院，郑州 450046

收稿日期:2021-09-02 出版日期:2022-06-23 发布日期:2022-06-25
通讯作者: 胡永浩，主要从事预防兽医学研究，E-mail: yhh0817@126.com
作者简介:李厚伟(1981-)，男，河南永城人，博士，主要从事动物免疫学研究，E-mail: lhw69877@163.com
基金资助:
国家自然科学基金面上项目(31772773；31972678)；河南省重大科技专项(181100110500)

Analysis of the Proliferation Characteristics of Porcine Deltacoronavirus on Suspension Cultured Porcine Kidney Cell LLC-PK1

LI Houwei¹, WANG Lei², ZHANG Xianfeng², HU Hui³, ZHANG Zhenzhen², ZHANG Yunfei³, LIU Lintao³, JI Xingyu², HU Yonghao^1*

1. College of Veterinary Medicine of Gansu Agricultural University, Lanzhou 730070, China;
2. Shangqiu Meilan Bioengineering Co., Ltd., Zhecheng 476200, China;
3. College of Veterinary Medicine of Henan Agricultural University, Zhengzhou 450046, China

Received:2021-09-02 Online:2022-06-23 Published:2022-06-25

摘要/Abstract

摘要： 旨在分析猪丁型冠状病毒(porcine deltacoronavirus，PDCoV)在悬浮培养的猪肾细胞LLC-PK1上的增殖特性，为PDCoV灭活疫苗的规模化生产提供细胞材料。采用逐步降血清法优化LLC-PK1细胞悬浮培养工艺；利用有限稀释法筛选PDCoV适应性细胞株；利用间接免疫荧光法鉴定PDCoV对LLC-PK1细胞的感染性；分别对PDCoV接种LLC-PK1悬浮细胞的初始密度、MOI、收毒时间、TPCK胰酶浓度等参数进行优化，确定最佳悬浮培养条件。成功筛选出可高效增殖PDCoV的单克隆悬浮细胞株LLC-PK1Sa，且利用其增殖的PDCoV可特异性的感染LLC-PK1细胞；PDCoV按MOI为10^-3接种于密度为2×10⁶ cells·mL^-1的LLC-PK1Sa细胞，当TPCK胰酶终浓度达到7.5 μg·mL^-1时，接毒后48 h收获的病毒液滴度最高。本研究首次实现了PDCoV在LLC-PK1Sa悬浮细胞中的高效增殖，并对悬浮培养条件进行了初步优化，可为PDCoV灭活疫苗的规模化生产提供理论参考。

关键词: LLC-PK1细胞, 悬浮培养, 猪丁型冠状病毒

Abstract: This study aimed to analyze the proliferation characteristics of porcine deltacoronavirus (PDCoV) in suspension cultured porcine kidney cells LLC-PK1, so as to provide Candidate cell for large-scale production of PDCoV inactivated vaccine. LLC-PK1 cells were suspended by gradually decreasing serum method. PDCoV adaptive monoclonal cell lines were screened by limited dilution method. Indirect immunofluorescence method was used to identify the infectivity of PDCoV. The initial cell density, MOI, time of receiving virus collection and TPCK pancreatin concentration were screened to determine the best suspension culture conditions. The suspension cell strain LLC-PK1Sa which can proliferate PDCoV efficiently was screened out; PDCoV can specifically infect LLC-PK1 cells; PDCoV inoculated LLC-PK1Sa cells with a density of 2×10⁶ cells·mL^-1 according to the MOI of 10^-3, When the final concentration of TPCK pancreatin reached 7.5 μg·mL^-1, the titer of virus solution harvested 48 h after inoculation was the highest. In this study, the efficient proliferation of PDCoV in LLC-PK1Sa suspension cells was realized for the first time, and the suspension culture conditions were preliminarily optimized, which could provide theoretical reference for large-scale production of PDCoV inactivated vaccine.

Key words: LLC-PK1 cells, suspension culture, PDCoV

中图分类号:

S852.659.6

李厚伟, 王蕾, 张先锋, 胡慧, 张真真, 张云飞, 刘林涛, 姬星宇, 胡永浩. 猪丁型冠状病毒在悬浮培养猪肾细胞LLC-PK1上的增殖特性分析[J]. 畜牧兽医学报, 2022, 53(6): 2024-2028.

LI Houwei, WANG Lei, ZHANG Xianfeng, HU Hui, ZHANG Zhenzhen, ZHANG Yunfei, LIU Lintao, JI Xingyu, HU Yonghao. Analysis of the Proliferation Characteristics of Porcine Deltacoronavirus on Suspension Cultured Porcine Kidney Cell LLC-PK1[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 2024-2028.

参考文献 11

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猪丁型冠状病毒在悬浮培养猪肾细胞LLC-PK1上的增殖特性分析

Analysis of the Proliferation Characteristics of Porcine Deltacoronavirus on Suspension Cultured Porcine Kidney Cell LLC-PK1

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