水痘-带状疱疹病毒糖蛋白I基因在昆虫细胞中的表达及纯化

目的 将北京水痘-带状疱疹病毒(VZV)84-7株克隆糖蛋白I(gpI)基因在杆状病毒-昆虫细胞表达系统中表达,并对其表达产物进行纯化。方法 采用PCR方法从VZVDNA中扩增gpI全基因序列,并将其插入杆状病毒转移质粒pBacPAK9中,获得重组转移质粒pBacVZVgpI,对pBacVZVgpI中的插入基因进行测序。重组转移质粒与线性杆状病毒BacPAK6DNA(Bsu36Idigested)共转染Sf9昆虫细胞,获得重组病毒BacPAK-gpI。通过亲和层析纯化重组蛋白,并检测其抗原性。结果 PCR扩增得到gpI基因,测序结果表明克隆的外源基因正确。经SDS-聚丙烯酰胺凝胶电泳(SDS—PAGE)、免疫印迹(weotem-blot)方法证明gpI基因在昆虫细胞中获得表达,表达产物在培养72h达到高峰,重组蛋白的相对分子质量约为58000和70000,与理论值相符,蛋白质加工与天然蛋白类似。动物实验结果表明,重组蛋白具有较好的免疫原性,可刺激小鼠产生中和抗体。SDS—PAGE检测纯化的重组蛋白,纯度达80％。纯化蛋白经western-blot和ELISA检测后显示,具有特异的抗体结合活性。结论 应用昆虫细胞表达水痘-带状疱疹病毒gpI基因,可为水痘-带状疱疹病毒抗原定量分析、糖蛋白ELISA的研制和制备亚单位疫苗提供基础。

Expression and purification of varicella-zoster virus glycoprotein I gene in insect cells

Objective To express the cloned gene glycoprotein I (gpI) of varicella-zoster virus (VZV), Beijing VZV 84-7 strain in insect cells and to purify its exprression product. Methods The gene coding for gpI of VZV was amplified from viral DNA by PCR and cloned into baculovirus transfer vector (pBacPAK9), and recombinent transfer vector plasmid pBacVZVgpI was obtained. The inserted gpI gene in the pBacVZVgpI was sequenced. Insect cells Sf 9 were co-transfected with the recombinant transfer vector plasmid pBacVZVgpI and wild type linear baculovirus BacPAK6 (digested with Bsu36I) DNA. The recombinant baculoviruses containing the VZV 84-7 gpI gene was isolated through several rounds of limited dilution. Recombinant protein gpI was expressed in insect cells Sf 9, postinfected with recombinant baculoviruses.The expressed recombinant gpI was purified by lectin affinity chromatography and its antigenicity and immunogenicity were investigated. Results The gene coding for gpI of VZV was obtained by PCR and the gpI gene of pBacPAK9 was confirmed by DNA sequencing. The recombinant gpI was expressed in insect cells Sf 9, post-infected with recombinant baculovirus and identified by SDS-PAGE and western blotting, with its product in cell culture reaching the peak in 72 hours and with a molecular mass of 58 kd and 70 kd, the same as theoretical values. Results of immunoassay with cell lysates infected by recombinant baculoviruses indicated that recombinant protein expressed in insect cells had ability of eliciting specific antibodies against native VZV in mice and complement-dependent neutralizing antibodies. The purified recombinant gpI gave a product with a purity of more than 80%. ELISA and western-blottiong analysis demonstrated that purified protein had specific VZV antibody-binding activity. This suggested that the recombinant gpI expressed in insect cells had the same biological characteristics as its native counterpart. Conclusion Baculovirus-insect cells could be used to express the gene of VZV gpI, which could provide a basis for quantitative analysis of VZV antigen, and preparation of its subunit vaccine.