论文部分内容阅读
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 (western 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 5 80 0 0和 70 0 0 0 ,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。
Objective To express gpI gene of 847 strains of VZV in baculovirus expression system and to purify its expression product. Methods The full-length gpI gene was amplified from VZVDNA by PCR and inserted into the baculovirus transfer plasmid pBacPAK9 to obtain the recombinant plasmid pBacVZVgpI. The inserted gene of pBacVZVgpI was sequenced. Recombinant transfer plasmids were co-transfected into Sf9 insect cells with the linear baculovirus BacPAK6 DNA (Bsu36Idigested) to obtain the recombinant virus BacPAK gpI. Recombinant proteins were purified by affinity chromatography and tested for antigenicity. Results The gpI gene was amplified by PCR. The sequencing results showed that the foreign gene was cloned correctly. The results of SDS PAGE and western blot showed that the gpI gene was expressed in insect cells and reached its peak at 72h after culture. The relative molecular mass of the recombinant protein was about 5 80 000 And 70 0 0 0, in line with the theoretical value, protein processing similar to the natural protein. The results of animal experiments show that the recombinant protein has good immunogenicity and can stimulate mice to produce neutralizing antibodies. Purified recombinant protein was detected by SDS PAGE with a purity of 80%. The purified protein detected by western blot and ELISA showed specific antibody binding activity. Conclusion The expression of varicella-zoster virus gpI gene in insect cells can provide a basis for the quantitative analysis of varicella-zoster virus antigens, the development of glycoprotein ELISA and the preparation of subunit vaccines.