稳定表达非洲猪瘟病毒P54蛋白的Vero细胞系的建立

doi:10.13560/j.cnki.biotech.bull.1985.2019-1099

生物技术通报 ›› 2020, Vol. 36 ›› Issue (5): 139-144.doi: 10.13560/j.cnki.biotech.bull.1985.2019-1099

稳定表达非洲猪瘟病毒P54蛋白的Vero细胞系的建立

王彩霞¹, 杜方原¹, 林祥梅¹, GrzegorzWozniakowski², 王勤¹, 冯春燕¹, 吴绍强¹

1.中国检验检疫科学研究院动物检疫研究所,北京 100176;
2.波兰国家兽医研究所猪病部,波兰普瓦维 24-100

收稿日期:2019-11-13 出版日期:2020-05-26 发布日期:2020-06-03
作者简介:王彩霞,女,硕士,研究方向：动物检疫;E-mail：wangcx@caiq.org.cn
基金资助:
“十三五”国家重点研发计划项目（2016YFD0501100）

Generation of a Vero Cell Line Stably Expressing African Swine Fever Virus P54 Protein

WANG Cai-xia¹, DU Fang-yuan¹, LIN Xiang-mei¹, Grzegorz Wozniakowski², WANG Qin¹, FENG Chun-yan¹, WU Shao-qiang¹

1. The Institute of Animal Quarantine,Chinese academy of Inspection and Quarantine,Beijing 100176;
2. Department of Swine Diseases,National Veterinary Research Institute,Puławy,Poland 24-100;

Received:2019-11-13 Published:2020-05-26 Online:2020-06-03

摘要/Abstract

摘要： 旨为建立稳定表达非洲猪瘟病毒（ASFV）P54蛋白的Vero细胞系,将ASFV-P54基因与绿色荧光基因Azami Green的融合基因片段,将其克隆至慢病毒载体pLV-puro中构建重组慢病毒质粒pLV-ASFV-P54-AG,将该质粒与慢病毒包装质粒pH1和pH2共转染HEK-293V细胞,包装表达ASFV-P54蛋白的慢病毒。将重组慢病毒在聚凝胺（Polybrene）的介导下感染Vero细胞,筛选出一株稳定表达ASFV-P54蛋白的Vero细胞系,命名为Vero-AG-ASFV-P54。间接免疫荧光试验表明,该细胞系能够与P54多克隆抗体反应;经波兰国家兽医研究所进一步验证,结果显示,该细胞系与ASFV抗体阳性血清也能发生反应,并且与阴性血清无反应。结果表明,Vero-AG-ASFV-P54细胞系能够稳定高效的表达具有生物活性的ASFV-P54蛋白。

关键词: 非洲猪瘟病毒, P54蛋白, Azami绿色荧光蛋白, Vero细胞, 慢病毒载体

Abstract: In order to establish the Vero cell lines that may stably express African swine fever virus（ASFV）,the fusion gene fragment from the ASFV-P54 gene with green fluorescent gene Azami Green was cloned into the lentivirus vector pLV-puro. The resulting plasmid,pLV-ASFV-P54-AG,was then co-transfected HEK-293V cells together with packaging plasmids pH1 and pH2 for packaging recombinant lentiviruses. The generated lentiviruses were then utilized to infect Vero cells mediated in the presence of polybrene. A Vero cell line,constitutively expressing the ASFV-P54 protein,was screened and designated as Vero-AG-ASFV-P54. Moreover,indirect immunofluorescence assays further demonstrated that the generated cell line reacted with P54 polyclonal antibody. The results further verified by National Veterinary Research Institute in Poland showed that the cell line reacted with the positive sera of ASFV antibody,but did not react with the negative sera. The above results reveal that the Vero-AG-ASFV-P54 cell line generated in this study may stably and efficiently express ASFV-P54 protein with biological activity.

Key words: African swine fever virus, P54, Azami Green, Vero cells, lentivirus vector

王彩霞, 杜方原, 林祥梅, GrzegorzWozniakowski, 王勤, 冯春燕, 吴绍强. 稳定表达非洲猪瘟病毒P54蛋白的Vero细胞系的建立[J]. 生物技术通报, 2020, 36(5): 139-144.

WANG Cai-xia, DU Fang-yuan, LIN Xiang-mei, Grzegorz Wozniakowski, WANG Qin, FENG Chun-yan, WU Shao-qiang. Generation of a Vero Cell Line Stably Expressing African Swine Fever Virus P54 Protein[J]. Biotechnology Bulletin, 2020, 36(5): 139-144.

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稳定表达非洲猪瘟病毒P54蛋白的Vero细胞系的建立

Generation of a Vero Cell Line Stably Expressing African Swine Fever Virus P54 Protein

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