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目的真核表达水痘-带状疱疹病毒(varicella-zoster virus,VZV)糖蛋白E胞外区(gE_(537)),并进行鉴定。方法用Oka株VZV(VZV-Oka)感染人二倍体细胞(2BS株),提取基因组DNA,以其为模板,PCR扩增gE_(537)目的片段,克隆至载体pCI-neo,构建重组真核表达质粒p CI-neo-g E_(537)-His。大量扩增后提取质粒,转染293FT细胞,瞬时表达,经镍柱纯化,获得目的蛋白gE_(537)-His,Western blot和ELISA法进行鉴定。结果经双酶切及测序鉴定,重组真核表达质粒pCIneo-g E_(537)-His构建成功。200 mmol/L咪唑洗脱液为洗脱目的蛋白的最佳咪唑浓度。纯化产物可与鼠抗gE糖蛋白单抗于相对分子质量约90 000处发生特异性结合,且可与mAb-10、mAb-12抗gE单抗发生反应。结论利用293FT细胞成功表达了gE_(537),为其免疫原性等相关研究奠定了基础。
Objective To eukaryotic expression of extracellular domain (gE_ (537)) of glycoprotein E of varicella-zoster virus (VZV). Methods Human diploid cells (2BS strain) were infected with VZV (OZV-Oka) strain. The genomic DNA was extracted and used as a template to amplify the target fragment of gE_ (537) and cloned into pCI-neo vector to construct recombinant Nuclear expression plasmid p CI-neo-g E 537-His. After amplification, the 293FT cells were transiently transfected into 293FT cells and purified by nickel column. The target protein gE_ (537) -His was obtained and identified by Western blot and ELISA. Results After double enzyme digestion and sequencing, the recombinant plasmid pCIneo-g E 537 -His was successfully constructed. 200 mmol / L imidazole eluate as the optimal concentration of imidazole protein. The purified product can specifically bind to the mouse anti-gE glycoprotein monoclonal antibody at a molecular weight of about 90,000 and can react with mAb-10 and mAb-12 anti-gE monoclonal antibody. Conclusion The 293F cells successfully expressed gE_ (537), which laid the foundation for their immunogenicity and other related research.