表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定

doi:10.11843/j.issn.0366-6964.2019.08.012

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (8): 1635-1641.doi: 10.11843/j.issn.0366-6964.2019.08.012

表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定

胡永新, 赵永刚, 张永强, 刘拂晓, 樊晓旭, 吴晓东*, 王志亮*

中国动物卫生与流行病学中心, 青岛 266032

收稿日期:2019-02-12 出版日期:2019-08-23 发布日期:2019-08-19
通讯作者: 王志亮,主要从事预防兽医学研究,E-mail:wangzhiliang@cahec.cn;吴晓东,主要从事预防兽医学研究,E-mail:wuxiaodong@cahec.cn
作者简介:胡永新(1987-),男,山东青岛人,助理研究员,博士,主要从事非洲猪瘟新型疫苗及诊断方法建立研究,Tel:0532-85639166,E-mail:huyongxin@cahec.cn
基金资助:
“十三五”国家重点研发计划（2017YFD0501800）；青岛市博士后应用研究项目

Construction and Identification of Replication Defective Recombinant Adenovirus Expressing ASFV P72 Protein

HU Yongxin, ZHAO Yonggang, ZHANG Yongqiang, LIU Fuxiao, FAN Xiaoxu, WU Xiaodong*, WANG Zhiliang*

China Animal Health and Epidemiology Center, National Research Center for Exotic Animal Disease, Qingdao 266032, China

Received:2019-02-12 Online:2019-08-23 Published:2019-08-19

摘要/Abstract

摘要： 本研究旨在构建能够表达非洲猪瘟病毒（ASFV） P72蛋白的缺陷型重组腺病毒。参考ASFV China-SY18株的基因序列，合成B646L基因。先将合成的B646L基因通过TOPO连接克隆到过渡载体pENTR/D-TOPO中；再将插入B646L基因的过渡载体pENTR/D-TOPO-ASFV-P72与pAd/CMV/V5-DEST腺病毒骨架载体进行重组，得到pAd-ASFV-P72重组质粒；重组质粒经PacⅠ酶切线性化后转染293A细胞，连续传代后获得重组腺病毒。结果表明，获得的Ad5-ASFV-P72重组腺病毒的滴度为2.53×10⁹ IFU·mL^-1；经免疫荧光方法及Western blot鉴定ASFV-P72蛋白在Vero细胞中成功表达，且能够与ASFV标准阳性血清发生特异性反应。本研究成功获得能够表达ASFV P72蛋白的重组腺病毒Ad5-ASFV-P72，不仅为ASFV多基因重组腺病毒载体疫苗的研制奠定了一定基础，还为ASFV相关血清学诊断方法的建立提供了安全有效的抗原。

关键词: 非洲猪瘟病毒, 复制缺陷型腺病毒, P72蛋白

Abstract: This study aimed to construct a defective recombinant adenovirus capable of expressing the P72 protein of African swine fever virus (ASFV). The ASFV B646L gene based on relevant China-SY18 isolate was synthesized. The synthetic B646L gene was cloned into transfer vector pENTR/D-TOPO. In order to construct a recombinant adenovirus, the recombination between the transfer vector pENTR/D-TOPO-ASFV-P72 and the backbone vector pAd/CMV/V5-DEST was done. The PacⅠ-linearized recombinant vector was transfected into 293A cell to produce recombinant adenovirus by serial passage. The results showed that the titers of the recombinant adenovirus Ad5-ASFV-P72 were 2.53×10⁹ IFU·mL^-1; the ASFV-P72 protein was expressed successfully in Vero cells by identification of the Indirect immunofluorescence assay and Western blot, and the expressed protein could reacted with ASFV standard positive serum specifically. Ad5-ASFV-P72 recombinant adenovirus was constructed successfully in the study. These findings not only lay the solid foundation for developing ASFV multi-gene recombinant adenoviral vector vaccine, but also provide a safe and effective antigen for the establishment serological diagnostic methods.

Key words: African swine fever virus, replication defective adenovirus, P72 protein

中图分类号:

S852.659.1

胡永新, 赵永刚, 张永强, 刘拂晓, 樊晓旭, 吴晓东, 王志亮. 表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定[J]. 畜牧兽医学报, 2019, 50(8): 1635-1641.

HU Yongxin, ZHAO Yonggang, ZHANG Yongqiang, LIU Fuxiao, FAN Xiaoxu, WU Xiaodong, WANG Zhiliang. Construction and Identification of Replication Defective Recombinant Adenovirus Expressing ASFV P72 Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1635-1641.

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表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定

Construction and Identification of Replication Defective Recombinant Adenovirus Expressing ASFV P72 Protein

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[1]	胡永新, 赵永刚, 张永强, 刘拂晓, 樊晓旭, 吴晓东, 王志亮. 表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定[J]. 畜牧兽医学报, 2019, 50(8): 1635-1641.
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[3]	龚振华，王丽萍，臧京帅，张康，刘春菊，吴晓东，张启迪，秦晓冰，陈琳琳，单虎，王树双，王志亮. 非洲猪瘟病毒p54蛋白的高效表达及在ELISA中的应用[J]. 畜牧兽医学报, 2013, 44(11): 1832-1837.