Xiong Sheng, Ren Xiang-Rong, Tang Yong-Hong, Su Kuan-Yuan, Yu Zhou-Yao, Luo Yong, Wang Yi-Fei, Li Jiu-Xiang
Biomedicine Research & Development Center, Jinan University, Guangzhou 510640, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Jan;19(1):19-23.
To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.
为了在巴斯德毕赤酵母中表达和分泌天然HBscFv（抗HBsAg单链Fv），从质粒pGEM-HBscFv中扩增HBscFv，然后亚克隆到表达载体pPICZalphaA中。将所得质粒pPIC-HBscFv线性化并转化到巴斯德毕赤酵母GS115中。通过直接PCR和对毕赤酵母转化子进行Zeocin抗性筛选鉴定出的重组毕赤酵母菌株，用甲醇进行培养和诱导。结果发现，由α因子引导的重组HBscFv可分泌到培养上清液中，水平达到80mg/L。通过间接ELISA证实了毕赤酵母产生的HBscFv的生物活性，这也表明培养上清液中HBscFv的生物活性在72小时达到峰值，并在诱导后期下降。PAS染色表明酵母产生的HBscFv是糖基化程度低或非糖基化的蛋白质。
