Cai Jian-piao, Qian Fei, Wang Jia-ying, Zhao Ying, Xu Xiao-jing, Jin Wei-rong, Che Xiao-yan
Clinical Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2010 Aug;44(8):721-5.
To achieve secretory and extracellular production of recombinant dengue virus serotypes I-IV envelope glycoprotein domain III (DENV-1-4 EDIII) in Pichia pastoris.
EDIII genes of DENVI-IV were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GS115. Clones highly produced in 4.0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni-NTA agarose beads were used for purification, while SDS-PAGE and Western blotting were used for identification.
The recombinant plasmids pPIC9K-DENV-1-4 EDIII were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant EDIII proteins were expressed in a secretory way with the molecular weight about 12 × 10(3) and specifically identified by anti-His monoclonal antibody and anti-DENVI-IV mice sera.
DENVI-IV EDIII proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.
在毕赤酵母中实现重组登革病毒血清型1 - 4包膜糖蛋白结构域III（DENV - 1 - 4 EDIII）的分泌型和胞外表达。
分别扩增登革病毒1 - 4型的EDIII基因，并克隆到载体pPIC9K中。然后将这些重组质粒线性化并转入毕赤酵母菌株GS115。对在4.0 mg/ml G418中高表达的克隆进行扩增，并用甲醇诱导以获得分泌型重组蛋白。使用Ni - NTA琼脂糖珠进行纯化，同时用SDS - PAGE和Western印迹进行鉴定。
构建了重组质粒pPIC9K - DENV - 1 - 4 EDIII，并成功转入毕赤酵母菌株GS115。重组EDIII蛋白以分泌方式表达，分子量约为12×10³，可被抗His单克隆抗体和抗登革病毒1 - 4型小鼠血清特异性识别。
从毕赤酵母表达系统中成功获得了登革病毒1 - 4型EDIII蛋白，可用于登革热疫苗、诊断试剂的研发以及E蛋白生物学功能的研究。
