中文摘要：

乙肝表面抗原结合蛋白（HBs Ag binding protein,SBP）是以HBs Ag为探针,通过人肝c DNA噬菌体表达库筛选的一种人源蛋白。SBP可特异性结合乙肝表面抗原（Hepatitis B virus surface antigen,HBs Ag）,增强乙肝疫苗的免疫效果,是一种潜在快速高效的免疫佐剂。成功构建了分泌型高表达SBP的毕赤酵母工程菌。对该菌株进行了放大规模发酵表达,并对纯化工艺进行了研究。在发酵实时检测过程中,自诱导剂甲醇加入开始,SBP蛋白的分泌表达量随时间推移而逐渐增加,发现于38h达到最佳水平且此时杂蛋白含量最少,是放罐收集菌液的最佳时间。18L发酵液在低温离心除菌体和沉淀物后可得到15L的上清液,再利用截留量为5k Da的超滤膜包将上清液浓缩至2L,将浓缩后的上清液依次通过S200分子筛柱和TDEAE阴离子交换柱进行分离纯化,可得到300ml浓度为1.125mg/ml、纯度达98%的目标蛋白液,发酵液得率为22.5mg/L;最后将蛋白液定量分装冻干低温保存。所获得的放大规模SBP发酵诱导表达条件和SBP蛋白的分离纯化工艺,为SBP蛋白大规模生产奠定了坚实的基础。

英文摘要：

The HBs Ag binding protein（ SBP） was screened out from human liver c DNA phage expression library with HBs Ag as a probe. SBP has the ability to enhance the immune response of Hepatitis B vaccine. It is proved to be a potential efficient immune adjuvant. The secretory SBP-expressing Pichia pastoris strains with high protein expression quantity has successfully constructed,then was fermented in the 18 L fermentor. The results showed that SBP could achieve the maximum expression after 38 h in methanol induction according to real time detecting; meanwhile hybrid proteins were the least so that it＇s the most appropriate time to collect bacteria liquid. 15 L supernatant could be obtained at low temperature centrifugation. Through the ultrafiltration membrane package whose interception was 5k Da,the supernatant was concentrated to about 2L. Then the concentrated fermentation supernatant was passed through sephadex S200 and the TDEAE column respectively. The attained300 ml SBP protein was with a purity of 98% and 1. 125 mg / ml as concentration. The SBP yield from fermented liquid was about 22. 5mg / L. Finally,the lyophilized proteins were preserved at low temperature. The work on the fermentation process optimized the time,identified the purification process and lay a solid foundation for the mass production of SBP.