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High-level production of a functional recombinant hepatitis B virus polymerase in insect cells with a baculovirus expression system

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Summary

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

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Authors and Affiliations

  1. Institute of Liver Diseases, Tongji Hospital, Tongii Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Wang Xiaoyan  (王晓燕), Gao Linlin  (高琳琳), Li Yan  (李岩) & Lin Jusheng  (林菊生)

  2. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430030, China

    Deng Fei  (邓菲) & Zhang Yanfang  (张艳芳)

Authors
  1. Wang Xiaoyan  (王晓燕)
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  2. Gao Linlin  (高琳琳)
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  3. Deng Fei  (邓菲)
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  4. Zhang Yanfang  (张艳芳)
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  5. Li Yan  (李岩)
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  6. Lin Jusheng  (林菊生)
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Corresponding author

Correspondence to Lin Jusheng  (林菊生).

Additional information

WANG Xiaoyan, female, born in 1980, M.D., Ph.D.

This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30330680).

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Wang, X., Gao, L., Deng, F. et al. High-level production of a functional recombinant hepatitis B virus polymerase in insect cells with a baculovirus expression system. J. Huazhong Univ. Sc. Technol. 27, 269–273 (2007). https://doi.org/10.1007/s11596-007-0313-9

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  • DOI: https://doi.org/10.1007/s11596-007-0313-9

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