猪瘟病毒C株表位突变毒株的构建及拯救

doi:10.11843/j.issn.0366-6964.2024.02.027

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (2): 698-705.doi: 10.11843/j.issn.0366-6964.2024.02.027

猪瘟病毒C株表位突变毒株的构建及拯救

刘元杰, 徐璐, 朱元源, 徐嫄, 张乾义, 李翠, 李明, 夏应菊, 王琴, 刘业兵, 赵启祖*, 邹兴启*

中国兽医药品监察所国家猪瘟参考实验室, 北京 100081

收稿日期:2023-03-27 出版日期:2024-02-23 发布日期:2024-02-27
通讯作者: 邹兴启,主要从事兽用生物制品检验和质量控制研究工作,E-mail:zouxingqi@163.com,Tel:010-62103670;赵启祖,主要从事分子病毒学与免疫学研究,E-mail:zhaoqizu@163.com
作者简介:刘元杰(1992-),男,河北保定人,硕士,主要从事兽用生物制品检验及研究,E-mail:12301675@qq.com,Tel:010-62103919
基金资助:
中国兽医药品监察所"兽药行业公益性重点专项"-猪瘟疫苗免疫与自然感染(野毒感染)鉴别诊断技术研究

The Construction and Rescue of Epitope Mutant Strain of Classical Swine Fever Virus C Strain

LIU Yuanjie, XU Lu, ZHU Yuanyuan, XU Yuan, ZHANG Qianyi, LI Cui, LI Ming, XIA Yingju, WANG Qin, LIU Yebing, ZHAO Qizu*, ZOU Xingqi*

National Swine Fever Reference Laboratory of China Institute of Veterinary Drug Control, Beijing 100081, China

Received:2023-03-27 Online:2024-02-23 Published:2024-02-27

摘要/Abstract

摘要： 本研究旨在突变猪瘟病毒C株WH303位点,构建感染性克隆,为猪瘟病毒C株标记疫苗的研究提供候选毒株。本试验以猪瘟病毒C株为研究材料,经重叠PCR方法突变WH303位点,再分别连接至C株和C-flag株全长感染性克隆。体外转录RNA,转染PK15细胞,细胞连续传代完成病毒的拯救和增殖。结果显示,经特异性限制内切酶酶切反应验证,感染性克隆构建成功。经RT-PCR、过氧化物酶单层细胞试验检测,表明病毒成功拯救。病毒细胞传代至17代,检测到病毒,表明病毒可以稳定传代。综上,本研究成功构建了C株WH303突变感染性克隆MC和MC-flag,并成功拯救MC株,得到可以稳定传代的病毒株。

关键词: 猪瘟病毒, C株, WH303位点, 突变, 感染性克隆

Abstract: An infectious clone was constructed by mutating the WH303 site of CSFV C strain to provide candidate strains for the study of marker vaccine of CSFV C strain. In this study, CSFV C strain was used as the research material, and WH303 site was mutated by overlapping PCR method, and then cs-full and CS-full-flag were cloned into the full-length infectious clones of CSFV C strain. In vitro transcription RNA, and then transfected into PK15 cells, continuous passage of cells to complete the cell rescue and proliferation. The results showed that the specific restriction endonuclease digestion reaction verified the successful construction of infectious clone. RT-PCR and peroxidase monolayer cell test detection indicated that the virus had been rescued successfully. The virus can still be detected in the 17th generation of virus cell, indicating that the virus can be stably passaged. In summary, MC and MC-flag of C strain WH303 mutated infectious clones were constructed successfully in this study, MC strain was saved successfully, and the stable virus strain was obtained.

Key words: swine fever virus, C strain, site WH303, mutation, infectious clone

中图分类号:

S852.651

刘元杰, 徐璐, 朱元源, 徐嫄, 张乾义, 李翠, 李明, 夏应菊, 王琴, 刘业兵, 赵启祖, 邹兴启. 猪瘟病毒C株表位突变毒株的构建及拯救[J]. 畜牧兽医学报, 2024, 55(2): 698-705.

LIU Yuanjie, XU Lu, ZHU Yuanyuan, XU Yuan, ZHANG Qianyi, LI Cui, LI Ming, XIA Yingju, WANG Qin, LIU Yebing, ZHAO Qizu, ZOU Xingqi. The Construction and Rescue of Epitope Mutant Strain of Classical Swine Fever Virus C Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 698-705.

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猪瘟病毒C株表位突变毒株的构建及拯救

The Construction and Rescue of Epitope Mutant Strain of Classical Swine Fever Virus C Strain

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