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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310OD600,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。
The prevalence of Hepatitis B virus poses a great threat to people’s life and health. Effective and accurate diagnosis and preventive vaccines are the main means to stop the epidemic. Hepatitis B surface antigen is the main component of diagnostic reagents and vaccines. In this study, the fermentation conditions of Pichia pastoris stably expressing HBsAg were studied. The effects of different media, dissolved oxygen, the concentration of inducer methanol and pH value on the growth and recombinant protein expression were studied by shake flask batch culture. In 10L fermenter by fed-batch culture method to study the expansion of the production of recombinant HBsAg. The results showed that FBS inorganic salt synthetic medium was the ideal industrial fermentation medium. Dissolved oxygen had a significant effect on the growth and expression of the bacterial cells. The best final concentration of methanol induced by methanol was 1% (V / V) The pH is 5.4 ~ 6.0. After the fermenter was expanded and cultured, the recombinant HBsAg was highly expressed by ELISA and SDS-PAGE. The final biomass reached 310 OD600 and the expression reached 27 mg / L. Electron microscopy of recombinant hepatitis B antigen expression can be self-assembled as 22nm class virus particles, for a new generation of HBV serological diagnosis and early diagnosis of large-scale production of vaccines provide a reference.