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2025年8月13日 星期三
农业生物技术学报  2021, Vol. 29 Issue (4): 780-788    DOI: 10.3969/j.issn.1674-7968.2021.04.016
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
稳定表达蓝舌病病毒VP6蛋白的BHK-21细胞系构建
李占鸿1, 宋子昂2, 杨振兴1, 李卓然1, 廖德芳1, 李华春1, 杨恒1,*
1 云南省畜牧兽医科学院 云南省热带亚热带动物病毒重点实验室,昆明 650224;
2 云南农业大学 动物医学院,昆明 650201
Construction of BHK-21 Cell Line Stably Expressing VP6 Protein of Bluetongue virus
LI Zhan-Hong1, SONG Zi-Ang2, YANG Zhen-Xing1, LI Zhuo-Ran1, LIAO De-Fang1, LI Hua-Chun1, YANG Heng1,*
1 Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming 650224, China;
2 College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China
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摘要 蓝舌病病毒(Bluetongue virus, BTV)的VP6蛋白(viral structural protein 6)在病毒复制与基因组组装中发挥着重要作用,构建稳定表达BTV VP6蛋白的细胞系,可为VP6蛋白功能研究及基因工程疫苗研制提供基础资料。本研究通过RT-PCR扩增BTV的VP6基因,分别构建出表达VP6蛋白的原核表达质粒pET32-BTV-VP6与真核表达质粒pCDH-BTV-VP6。将pET32-BTV-VP6转入大肠杆菌(Escherichia coli),在16 ℃进行诱导培养,VP6重组蛋白部分以可溶形式表达,表达量约为总菌体蛋白的24.5%。采用镍离子亲和层析对表达的VP6重组蛋白进行纯化并免疫新西兰兔(Oryctolagus cuniculu),制备抗VP6蛋白的多克隆抗体,抗体效价可达1∶12 000;Western blot与免疫荧光染色证实,制备的多克隆抗体可与BTV感染细胞产生的VP6蛋白发生特异性结合。将真核表达质粒pCDH-BTV-VP6转染BHK-21细胞,使用嘌呤霉素对转染后的细胞进行连续筛选,构建稳定表达BTV VP6蛋白的细胞系BHK-BTV-VP6;以兔抗VP6多克隆抗体为一抗,进行Western blot与免疫荧光检测,证实VP6蛋白在细胞中稳定表达。本研究实现了BTV VP6重组蛋白在大肠杆菌中的成功表达与纯化,构建了稳定表达BTV VP6蛋白的细胞系,为BTV VP6蛋白功能研究以及新型基因工程疫苗研发提供物质基础。
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李占鸿
宋子昂
杨振兴
李卓然
廖德芳
李华春
杨恒
关键词 蓝舌病病毒(BTV)VP6蛋白原核表达细胞系    
Abstract:VP6 protein (viral structural protein 6) of Bluetongue virus (BTV) plays an important role in virus replication and genome assembly. Developing cell line stably expressing the BTV VP6 protein will provide foundation for the research of VP6 protein function and genetic engineering vaccine. In present study, VP6 gene of BTV was amplified by RT-PCR to construct prokaryotic expression plasmid of pET32-BTV-VP6 and eukaryotic expression plasmid of pCDH-BTV-VP6. The pET32-BTV-VP6 was transferred into Escherichia coli and recombinant VP6 protein was expressed in soluble form at 16 ℃, which accounted for 24.5% of total bacterial protein. The recombinant VP6 protein was purified using Ni2+-NTA affinity chromatography and was used to immunize rabbits (Oryctolagus cuniculu) for preparation of polyclonal antibodies against VP6 protein of BTV. The antibody titer of the immunized rabbits reached 1∶12 000 as confirmed by indirect ELISA. VP6 protein expressed by BTV-infected cells was detected by Western blot and indirect immunofluorescence assay (IFA) using the prepared serum against VP6. To construct BHK-BTV-VP6 cell line which stably expressed VP6 protein of BTV, pCDH-BTV-VP6 was transfected into BHK-21 cells and continuously screened by puromycin. The stable expression of VP6 protein in the BHK-BTV-VP6 cell line was confirmed by Western blot and IFA using rabbit polyclonal antibody against BTV VP6 as primary antibody. Conclusively, the present study successfully expressed and purified BTV VP6 recombinant protein in E. coli, and constructed a cell line stably expressing BTV VP6 protein, which provided material basis for the research on VP6 protein function and the development of new genetic engineering vaccine.
Key wordsBluetongue virus (BTV)    VP6 protein    Prokaryotic expression    Cell line
收稿日期: 2020-07-13      出版日期: 2021-04-01
ZTFLH:  S852.65+9.4  
基金资助:国家自然科学基金(31760744); 国家重点研发计划(2016YFD0500908); 云南省中青年学术和技术带头人后备人才培养项目(2017HB055)
通讯作者: * yangheng2008.cool@163.com   
引用本文:   
李占鸿, 宋子昂, 杨振兴, 李卓然, 廖德芳, 李华春, 杨恒. 稳定表达蓝舌病病毒VP6蛋白的BHK-21细胞系构建[J]. 农业生物技术学报, 2021, 29(4): 780-788.
LI Zhan-Hong, SONG Zi-Ang, YANG Zhen-Xing, LI Zhuo-Ran, LIAO De-Fang, LI Hua-Chun, YANG Heng. Construction of BHK-21 Cell Line Stably Expressing VP6 Protein of Bluetongue virus. 农业生物技术学报, 2021, 29(4): 780-788.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2021.04.016     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2021/V29/I4/780
 
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