摘要
目的在CHO细胞中稳定表达水痘-带状疱疹病毒(VZV)糖蛋白E(gE)。方法PCR扩增VZVgE完整胞外结构域编码基因,克隆至哺乳动物细胞表达质粒PSGHVO中,与DHFR选择性基因和脂质体共转染CHO/dhfr-细胞,筛选稳定分泌人生长激素(hGH)和gE融合蛋白的细胞株。采用ELISA和Western blot方法检测细胞培养上清中hGH和gE融合蛋白的表达,并用Ni2+-NTA柱进行纯化。结果gE基因PCR扩增产物和重组表达质粒PSGHVO-gE的双酶切产物经琼脂糖凝胶电泳,均可见1500bp的目的基因条带。转染PSGHVO-gE质粒和DHFR基因的细胞培养上清液经ELISA和Western blot,确认有重组蛋白表达,gE表达量约为20mg/L;纯化后蛋白纯度约为90%。结论已在CHO细胞中稳定表达了VZVgE蛋白,为制备VZV表面抗原奠定了基础。
Objective To stably express the glycoprotein E（gE） of varicella-zoster virus （VZV） in CHO cells. Methods Amplify the fidl-length gene encoding extracellular domain of gE of VZV by PCR and insert into vector PSGHVO. Co-transfect CHO cells with the constructed recombinant plasmid PSGHVO-gE and DHFR gene as a selection marker and screen the cell strains stably secreting human growth hormone （hGH）-gE fusion protein. Determine the expressed fusion protein in cell culture superuatant by ELISA, identify by Western blot and purify by Ni^2＋-NTA column chromatography. Results The target gene fragment at a length of 1 500 bp was observed on both the agarose gel electrophoretic profiles of PCR products of amplified gE gene and constructed recombinant plasmid PSGHVO-gE. Both ELISA and Western blot proved the expression of recombinant hGH-gE fusion protein in culture superuatant of CHO cells co-transfected with recombinant plasmid PSGHVO-gE and DHFR gene. The gE reached an expression level of about 20 mg/L and a purity of 90% after purification. Conclusion The gE of VZV was stably expressed in CHO cells, which laid a foundation of preparation of VZV surface antigen.
出处
《中国生物制品学杂志》
CAS
CSCD
2008年第8期654-657,共4页
Chinese Journal of Biologicals
