水痘-带状疱疹病毒糖蛋白E在昆虫细胞中的表达、鉴定及免疫原性分析

目的:利用昆虫-杆状表达系统建立表达和纯化分泌形式的水痘-带状疱疹病毒(varicellazoster virus,VZV)糖蛋白gE的方法,并鉴定其理化性质及免疫原性。方法:利用Gibson assembly同源重组试剂盒快速构建重组质粒pFastbac-VZV gE。在sf9昆虫细胞中鉴定序列优化前后表达量的差异,并在High FiveTM细胞中大量表达。通过Ni-NTA亲和层析方法得到高纯度gE蛋白,之后通过酶联免疫吸附试验等验证了其理化性质,并进行小鼠免疫分析其免疫原性。结果:构建了pFastbac-VZV g E1/2重组质粒,经过PCR及双酶切鉴定后均为阳性克隆。使用BAC/PAC试剂盒提取Bacmid转染sf9细胞制备杆状病毒,经Western blot检测,sf9细胞开始表达gE蛋白且随着病毒代次升高g E蛋白表达量增加。序列优化后表达量明显增加,但是大部分以胞内形式存在。通过Ni-NTA一步亲和层析获得纯度较高的gE蛋白,并与VZV单抗9C8具有较好的反应性。通过免疫小鼠产生高滴度抗体,且免疫荧光结果显示其血清可以与天然病毒上的gE抗原结合。结论:成功获得了杆状表达系统表达的gE蛋白并且纯化和鉴定了蛋白质的性质及免疫原性,为进一步研发具有自主产权的VZV亚单位疫苗研究奠定了基础。

Expression and Identification of Varicella-Zoster Virus Glycoprotein E and Immunogenicity Assay

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.