摘要
目的在毕赤酵母中表达并纯化人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)辅助调节蛋白Vpu。方法从质粒p CDNA3.1-vphu中PCR扩增vphu基因,插入毕赤酵母表达载体p PICZαA,构建重组表达质粒p PICZαA-vphu,转化巴斯德毕赤酵母野生型X33菌株,经甲醇诱导表达带有6×His标签的重组Vpu蛋白。表达产物经Ni-Agarose 6×His标签纯化柱纯化。表达和纯化产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒p PICZαA-vphu经双酶切和测序鉴定证明构建正确;表达的重组蛋白相对分子质量约16 000,主要存在于细胞内;纯化后杂蛋白减少,与抗VPU抗体和抗His标签抗体均可结合,浓度为224μg/ml。结论利用毕赤酵母表达系统成功表达并纯化了HIV-1 Vpu蛋白,纯化的蛋白具有良好的抗原活性,为进一步研究其结构和功能以及靶点药物筛选模型的建立奠定了基础。
Objective To express the Vpu protein of human immunodeficiency virus type 1 （HIV-1） in Pichia pastoris and purify the expressed product. Methods The vphu gene was amplified from plasmid pCDNA3. 1-vphu by PCR and inserted into expression vector pPICZaA. The constructed recombinant plasmid pPICZaA-vphu was transformed to wild type P. postoris X33 strain and induced with methanol. The expressed 6 x His-tagged Vpu protein was purified by Ni- Agarose column chromatography. The expressed and purified proteins were identified by SDS-PAGE and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pPICZaA-vphu was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 16 000, mainly existed within the cells, in which the foreign protein decreased after purification. The purified protein showed binding to both antibodies against VPU and His tag, of which the concentration was 224 μg/ml. Conclusion HIV-1 Vpu protein with good antigenic activity was successfully expressed and purified, which laid a foundation of further study on the structure and function of HIV-1 Vpu and establishment of model for screening of target drugs.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第4期339-343,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(81202976)
北京市自然科学基金(7142013)
关键词
人类免疫缺陷病毒1型
Vpu蛋白
毕赤酵母
表达
纯化
Human immunodeficiency virus type 1 （HIV-1）
Vpu protein
Pichia pastoris
Expression
Purification
