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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (10): 33-41    DOI: 10.13523/j.cb.2105047
技术与方法     
利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*
朱潇静1,王芮1,张欣欣1,靳家鑫1,路闻龙1,丁大顺1,霍翠梅2,李青梅3,孙爱军1,**,庄国庆1,**()
1 河南农业大学动物医学院 国家动物免疫学国际联合研究中心 郑州 450002
2 济宁市畜牧业发展中心 济宁 272004
3 河南省农业科学院 郑州 450002
Construction of MDV Recombinant Vaccine Strain Integrated F Gene Using Bacterial Artificial Chromosome Technique
ZHU Xiao-jing1,WANG Rui1,ZHANG Xin-xin1,JIN Jia-xin1,LU Wen-long1,DING Da-shun1,HUO Cui-mei2,LI Qing-mei3,SUN Ai-jun1,**,ZHUANG Guo-qing1,**()
1 International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China
2 Jining City Animal Husbandry Development Center, Jining 272004, China
3 Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
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摘要：

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。
关键词： 细菌人工染色体;  新城疫病毒;  F基因;  马立克氏病毒;  重组疫苗    
Abstract:

Objective: Marek’s disease virus (MDV) is an alphaherpesvirus and is classified into three serotypes: MDV serotype 1 (MDV-1) which includes the pathogenic strains and their derivatives; MDV serotype 2 (MDV-2) which consists of non-oncogenic viruses; and MDV serotype 3 (MDV-3), which is also referred to as turkey herpesvirus 1 (HVT). All three MDV serotypes have been used as vaccines. CVI988 is a cell culture passage attenuated MDV-1 virus and the gold standard among MD vaccines. CVI988 could be used as a viral vector to express exogenous gene. MDV and Newcastle disease virus (NDV) co-infection in chickens is very popular in the field, but there are few candidates of recombinant vaccines to prevent the infection of both viruses at the same time. Methods: In this study, CVI988 expressing F gene was constructed by the bacterial artificial chromosome (BAC) technology and packaged into recombinant virus. After it was amplified by PCR, the F gene together with Kana gene fragment was integrated into CVI988 BAC by homologous recombination to generate CVI988 BAC-F. Subsequently, CVI988 BAC-F was transfected into chicken embryo fibroblast (CEF) cells by calcium phosphate to rescue recombinant virus, which was confirmed by Western blotting and indirect immunofluorescence assay (IFA) assays. Results: The results of virus growth curve and plaque area measurement showed that the insertion of F gene did not affect virus proliferation in vitro. Conclusion: We successfully constructed a recombinant CVI988 BAC virus, which provides a basis for development of novel vaccines to prevent and control co-infection of NDV and MDV.
Key words: Bacterial artificial chromosome    Newcastle disease virus    F genes    Marek’s disease virus    Recombinant vaccines
收稿日期: 2021-05-25 出版日期: 2021-11-08
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(31802160);河南省留学人员科研择优项目(30602136);河南省高等学校重点科研项目(22A230011)
通讯作者: 孙爱军,庄国庆     E-mail: gqzhuang2008@163.com
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朱潇静
王芮
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路闻龙
丁大顺
霍翠梅
李青梅
孙爱军
庄国庆

引用本文:

朱潇静,王芮,张欣欣,靳家鑫,路闻龙,丁大顺,霍翠梅,李青梅,孙爱军,庄国庆. 利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*[J]. 中国生物工程杂志, 2021, 41(10): 33-41.

ZHU Xiao-jing,WANG Rui,ZHANG Xin-xin,JIN Jia-xin,LU Wen-long,DING Da-shun,HUO Cui-mei,LI Qing-mei,SUN Ai-jun,ZHUANG Guo-qing. Construction of MDV Recombinant Vaccine Strain Integrated F Gene Using Bacterial Artificial Chromosome Technique. China Biotechnology, 2021, 41(10): 33-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2105047    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I10/33

名称 引物基因序列(5'-3')
P1-F CGATATACATTTGCTTATTTCGCTGTCCACCCATAACCGAGAGTTGACATTGATTATTG
P1-R CATCTATACTATTATAACATACTTGTATCATTAAGTTCCATACCATAGAGCCCACCGCATC
P2-F GTAGATTCTTCTGGTTCG
P2-R CTGTGAAAATTTAT CCC
表1  PCR引物序列
名称 引物基因序列(5'-3')
OVO-F CACTGCCACTGGGCTCTGT
OVO-R GCAATGGCAATAAACCTCCAA
ICP4-F TTATTGCCCCGTACTCACCG
ICP4-R CATTTAAAGTCTTTCATGCCAAAC
表2  qPCR引物序列
图1  F-Kana基因PCR扩增结果
图2  PCR鉴定电转化结果
图3  去除Kana基因PCR鉴定结果
图4  重组病毒CVI988 BAC-F表达鉴定结果
图5  重组病毒CVI988 BAC-F的体外增殖比较
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