Wei sheng wu xue bao = Acta microbiologica Sinica · 2014
[Construction of baculovirus vector with WPRE regulatory element to express Newcastle disease virus F gene in primary chicken embryo cells].
Аннотация на английскомСтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Gao Dongni, Ping Wenxiang, Jin Liying, Shen Wanli, Song Gang, Tang Xiaoyan, An Qi, Shen Yan, Ge Jingping
Аннотация
OBJECTIVE: To construct the recombinant baculovirus with mammaliancell-specific promoter and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), to highly express Newcastle disease virus (NDV) F gene in the primary chicken embryo cells.
METHOD: We extracted total RNAs from NDV La Sota strain. Then the F gene was amplified by reverse transcription polymerase chain reaction. We constructed the baculoviral vector (pCMV-WPRE-F) with F gene fused with the WPRE near its 3'end, which expressed under the control of the CMV promoter. The F gene recombinant bacmid was obtained by Bac-to-Bac system and transfected into sf9 insect cells to acquire F gene recombinant baculovirus. After amplification of recombinant baculovirus, the recombinant virus was transfected into chicken primary cells with 50 multiplicity of infection, and the proteins were harvested at 72 h after infection. The F protein expression levels mediated by WPRE regulatory element were analyzed.
RESULTS: Western blot results show that the F gene was successfully expressed in chicken primary cells. The product was a 56kDa protein and could be recognized by anti-NDV serum. The WPRE fusion significantly improved the F gene expression as 10 mmol/L butyrate did, but different to butyrate, the WPRE regulatory element was nontoxic to cells.
CONCLUSION: The optimized recombinant baculovirus could efficiently deliver NDV F gene into chicken primary cells and express the F antigen protein. In addition, the WPRE regulatory element could increase the expression levels of exogenous gene mediated by baculovirus in chicken primary cells. The research provides us a potential basis for the gene engineered vaccines of NDV and other avian infectious disease based on baculovirus vector.
METHOD: We extracted total RNAs from NDV La Sota strain. Then the F gene was amplified by reverse transcription polymerase chain reaction. We constructed the baculoviral vector (pCMV-WPRE-F) with F gene fused with the WPRE near its 3'end, which expressed under the control of the CMV promoter. The F gene recombinant bacmid was obtained by Bac-to-Bac system and transfected into sf9 insect cells to acquire F gene recombinant baculovirus. After amplification of recombinant baculovirus, the recombinant virus was transfected into chicken primary cells with 50 multiplicity of infection, and the proteins were harvested at 72 h after infection. The F protein expression levels mediated by WPRE regulatory element were analyzed.
RESULTS: Western blot results show that the F gene was successfully expressed in chicken primary cells. The product was a 56kDa protein and could be recognized by anti-NDV serum. The WPRE fusion significantly improved the F gene expression as 10 mmol/L butyrate did, but different to butyrate, the WPRE regulatory element was nontoxic to cells.
CONCLUSION: The optimized recombinant baculovirus could efficiently deliver NDV F gene into chicken primary cells and express the F antigen protein. In addition, the WPRE regulatory element could increase the expression levels of exogenous gene mediated by baculovirus in chicken primary cells. The research provides us a potential basis for the gene engineered vaccines of NDV and other avian infectious disease based on baculovirus vector.