Liu Rushi, Lin Qinlu, Sun Yi, Qiu Yilan, Xu Tian, Ding Hai, Guo Xiangrong
Colleges of Life Sciences, Hunan Normal University, Key Laboratory of Microbial Molecular of Hunan Province, Chnagsha 410081, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Dec;24(12):2098-105.
Hepatitis B virus (HBV) infection can cause the severe threat to the health of the people around the world. It depends upon the development of efficient diagnostic reagent and vaccine to prevent the prevalence of HB. In this study, we constructed the high expression recombinant Pichia pastoris and performed the screening tests in shake flasks to obtain the optimal values of several key fermentation parameters. Based on their effects on the growth and expression level of recombinant strains, FBS was the optimal industrial medium. The optimal values for the dissolve oxygen (DO), the final concentrations of methanol and the pH values were 50 mL, 1% (V/V) and 5.4-6.0, respectively. The optimal values of the parameters simulated in shake flasks were successfully scaled up to 10 L bioreactors to achieve high-throughput production: 310 OD600 in biomass and 27 mg/L in recombinant HBsAg. The expressed recombinant HBsAg in P. Pastoris was confirmed by SDS-PAGE and Western blotting. Electron microscopy examination showed that the purified protein could be self-assembled to 22 nm virus-like particles. The results provided a basis for industrial scale-up production of diagnostic reagent and vaccine of next generation against HB.
乙型肝炎病毒（HBV）感染对全球人民的健康构成严重威胁。预防乙肝流行依赖于高效诊断试剂和疫苗的研发。本研究构建了高表达重组毕赤酵母，并在摇瓶中进行筛选试验以获得几个关键发酵参数的最佳值。基于其对重组菌株生长和表达水平的影响，胎牛血清（FBS）是最佳工业培养基。溶解氧（DO）、甲醇终浓度和pH值的最佳值分别为50 mL、1%（V/V）和5.4 - 6.0。摇瓶中模拟的参数最佳值成功放大至10 L生物反应器以实现高通量生产：生物量为310 OD600，重组乙肝表面抗原（HBsAg）为27 mg/L。通过SDS - PAGE和Western印迹法证实了毕赤酵母中表达的重组HBsAg。电子显微镜检查表明，纯化后的蛋白可自组装成22 nm病毒样颗粒。这些结果为下一代乙肝诊断试剂和疫苗的工业化放大生产提供了依据。
