目的真核细胞稳定表达水痘-带状疱疹病毒（VZV）糖蛋白E（g E）的胞外域基因,应用g E抗原建立VZV感染的血清学试验来评价VZV疫苗的免疫效果。方法构建含有VZV g E胞外域基因的真核表达质粒p CDNA3.1-g E,测序后脂质体转染COS-7细胞,经G418筛选出稳定表达VZV g E的细胞株。采用RT-PCR法检测VZV g E的mRNA,Western blot和间接免疫荧光法检测g E的免疫反应性,表达产物经Ni2＋-NTA柱纯化后包被ELISA板,对127份0~10岁正常儿童血清中VZV-Ig G抗体水平进行检测。结果成功筛选出能够稳定表达VZV g E胞外域基因的COS-7细胞株,RT-PCR检测到g E的mRNA,经Western blot和间接免疫荧光鉴定,g E具有明显的免疫反应性,COS-7细胞株和其培养上清液中均有g E融合蛋白,表达量约为0.632 mg/ml,纯度约为90%。ELISA实验检测了127份0~10岁儿童血清中VZV-Ig G抗体,总阳性率为81.89%,特异度和灵敏度分别为93.75%和88.24%。结论本实验获得稳定高效表达VZV g E胞外域基因的COS-7细胞株,建立的ELISA血清学检测方法有助于VZV感染的流行病学研究和对易感人群VZV感染的诊断和预防。
Objective To express varicella-zoster virus （VZV） glycoprotein E （gE）extracellular domain in eukary- otic cells, and then use it to develop a serological test for diagnosis of VZV infection as well as for evaluation of VZV vaccination efficiency. Methods Constructing eukaryotic expression plasmid pCDNA3.1-gE,which contained coding gene for the extracellular domain of VZV gE. The authenticity of the insert was confirmed by DNA sequen- cing before it was transfected into COS-7 ceils. G418 was used to select the cell line that stably expressed VZV gE fusion protein. VZV gE gene expression in those cells was recognized by RT-PCR, and corresponding protein ex- pression was demonstrated by Western blot and immunofluorescence. An ELISA assay was developed with the use of Ni2~ -NTA column purified gE as the coating antigen, and the assay was then used to detect IgG antibodies against VZV in 127 serum specimens of normal children. Results COS-7 cell lines that stably expressed VZV gE extracel- lular domain protein was cloned successfully, mRNA encoding gE was determined by RT-PCR. The gE has obvious immunoreactivity detected by Western blot and indirect immunofluorescence. Western blot proved the expression of recombinant gE fusion protein in both ceils and culture supernatant of the COS-7 cells. The total gE protein was 0. 632 mg/ml and the purity was nearly 90%. The serological test detected VZV-IgG antibody in 81.89% of the 127 serum specimens from normal children. The specificity and the sensitivity of this ELISA test were 93.75% and 88.24% respectively. Conclusion COS-7 cell lines that efficiently express gE extracellular domain protein was obtained, and an ELISA based serology test was developed and validated. The test is not only beneficial to the epi- demiological studies of VZV infection, but also to the screen individuals that are at risk of VZV infection.
Acta Universitis Medicinalis Anhui