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
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.
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.
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