毕赤酵母发酵生产颗粒性乙肝表面抗原的条件优化(英文)  

Optimization of Fermentation Conditions for Maximal Recombinant Hepatitis B Surface Antigen Particle Production in Pichia pastoris

作　　者：刘如石[1] 林亲录[2] 孙意[3] 邱义兰[1] 徐甜[1] 丁海[1] 郭向荣[1] 

机构地区：[1]湖南师范大学生命科学学院微生物分子生物学湖南省重点实验室,长沙410081 [2]中南林业科技大学食品科学与工程学院,长沙410078 [3]中南大学湘雅二医院,长沙410004

出　　处：《生物工程学报》2008年第12期2098-2105,共8页Chinese Journal of Biotechnology

基　　金：Hunan Provincial Natural Science Foundation of China(Nos.08JJ3082,07JJ5048);Project of Hunan Science and Technology Commission(No.08kj0018)~~

摘　　要：乙型肝炎病毒的流行对人们的生命健康造成了极大的威胁，而有效准确的诊断和预防性疫苗是阻止其流行的主要手段，乙肝表面抗原是诊断试剂和疫苗的主要成分。本试验在构建稳定表达HBsAg的毕赤酵母菌株后，对其发酵条件进行了研究。采用摇瓶分批培养方法，探讨了不同培养基、溶解氧、诱导物甲醇的浓度以及pH值等因素对菌体生长与重组蛋白表达的影响。在10L发酵罐上采用分批补料培养的方法研究了进行扩大培养生产重组HBsAg。结果表明，FBS无机盐合成培养基是理想的工业发酵培养基，溶解氧对菌体的生长与表达有显著的影响，甲醇诱导最佳终浓度为1％（V／V），发酵的最适pH值为5．4～6．0。发酵罐放大培养后，ELISA和SDS—PAGE分析表明重组HBsAg获得了高效表达，最终菌体生物量达到310 OD600，表达量达到27mg／L。电子显微镜观察表达重组乙肝抗原可以自组装为22nm类病毒颗粒，为HBV的新一代早期血清学诊断和疫苗的大规模生产提供了一定的参考。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.

关 键 词：发酵参数 高表达 优化筛选 毕赤酵母 重组乙肝表面抗原 

分 类 号：R392[医药卫生—免疫学]