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| Expression and Identification of Varicella-Zoster Virus Glycoprotein E and Immunogenicity Assay |
QI Jia-long1,GAO Rui-yu1,JIN Shu-mei2,GAO Fu-lan1,YANG Xu1,MA Yan-bing1,LIU Cun-bao1,**( ) |
1 Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650000, China
2 Yunnan Institute of Materia Medica, Yunnan Baiyao Group Innovation and R&D Center, Yunnan Province Company Key Laboratory for TCM and Ethnic Drug of New Drug Creation, Kunming 650111, China; |
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Abstract Object: To establish an efficient Bac-to-Bac baculovirus expression system for the expression and purification of the secreted form of varicella-zoster virus (VZV) glycoprotein E (gE) and to evaluate the physical-chemical properties and immunogenicity of gE. Methods: Gibson assembly homologous recombination kit was used for pFastbac-VZV gE recombinant blasmid construction. Expression of the recombinant protein was prepared in baculovirus-infected High-Five TM insect cell after protein expression difference were identified between sequence optimization or non-optimization using sf9 insect cell. ELISA and Western blot were employed for the verification of physicochemical properties of Ni-NTA-exclusion chromatography purified protein gE. Immunogenicity assay of recombinant VZV gE was identified with serum titer determination and immunofluorescence reaction with na?ve VZV. Results: The pFastbac-VZV gE recombinant plasmid was successfully constructed according to PCR and enzyme digestion identification assay. Recombinant bacmids extracted with a BAC/PAC DNA extraction kit were transfected into sf9 insect cell for baculovirus preparation. VZV gE was expressed in baculovirus-infected sf9 cell in a generation-dependent increasing manner according to Western blot assay. Obviously, sequence optimization enhanced gE production, but most of the proteins existed intracellularly. Highly purified gE worked well with VZV monoclone antibody 9C8 by ELISA. High reaction titer of immune serum with gE was identified and immunofluorescence showed interaction between serum and VZV in APER-19 cell. Conclusion: VZV gE recombinant protein highly expressed in Bac-to-Bac baculovirus expression system are generated and laid the foundation for further research and development of VZV subunit vaccine.
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Received: 04 January 2019
Published: 18 September 2019
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Corresponding Authors:
Cun-bao LIU
E-mail: Cunbao_Liu@163.com
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