Yuan Gai-Ling, Cai Xue-Peng, Jing Zhi-Zhong, Zheng Ya-Dong, Luo Xue-Nong, Jia Wan-Zhong, Li Hui, Ding Jun-Tao
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Jul;21(4):563-7.
TSO18 gene was subcloned into the Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-TSO18 was transformed into P. pastoris GS115 by electroporation so that the plasmid will be integrated with chromosome of P. pastoris. The P. pastoris strains containing multi-copy recombinant were screened by G418 and induced by methanol. The expression product was analyzed by SDS-PAGE, Western blot, deglycosylation, and purified by Sephadex column, and was used to immunize mice. The results indicated that the target protein was efficiently expressed in P. pastoris, and glycosylated moderately, and had immunological activity. In a 5 liter fermentor, the expression level of the target protein was up to 2.54 mg/mL. These results will benefit for the development of genetically engineering vaccine.
TSO18基因被亚克隆到毕赤酵母表达载体pPIC9K中。通过电穿孔将重组质粒pPIC9K - TSO18转化到毕赤酵母GS115中,使该质粒与毕赤酵母染色体整合。通过G418筛选含多拷贝重组体的毕赤酵母菌株,并用甲醇诱导。通过SDS - PAGE、Western印迹、去糖基化分析表达产物,并用葡聚糖凝胶柱纯化,用于免疫小鼠。结果表明,目标蛋白在毕赤酵母中高效表达,糖基化程度适中,具有免疫活性。在5升发酵罐中,目标蛋白的表达水平高达2.54 mg/mL。这些结果将有利于基因工程疫苗的开发。