毕赤酵母表达乙型肝炎病毒preS2S蛋白的纯化和免疫效果  

Purification and Immune Effect of Hepatitis B Virus preS2(epi)S Protein Expressed in Pichia pastoris

作　　者：李利 金立杰[1] 赵小琳[1] 杨屹[1] 刘岩松[1] 董厉子[1] 郝富勇[1] 李建平[1] 

机构地区：[1]长春生物制品研究所疫苗一室,长春130062

出　　处：《中国生物制品学杂志》2007年第4期296-299,共4页Chinese Journal of Biologicals

摘　　要：目的研究重组毕赤酵母表达的乙型肝炎病毒preS2S蛋白的纯化方法及免疫效果。方法甲醇诱导含preS2S基因的重组毕赤酵母菌,采用蔗糖区带速率离心、Butyl-S-Sepharose 4FF疏水层析和DEAE-Sepharose Fast Flow离子交换层析等方法,对表达产物进行纯化。纯化后蛋白经SDS-PAGE和Western blot方法鉴定,制备成疫苗,并进行效力试验。放射免疫法检测免疫小鼠血清抗体浓度,计算ED50和相对效力。ELISA法检测血清中的preS2S抗体。结果纯化后的preS2(epi)S蛋白相对分子质量约为27000,且能与特异性抗体结合。两批纯化蛋白制备的疫苗ED50分别为0.35和0.48μg/ml,参比苗为0.52μg/ml。2批疫苗的体外相对效力分别为1.5和1.1,均合格,血清抗体平均浓度为808和784mIU/ml,参比苗为312mIU/ml。2批疫苗免疫小鼠后均产生了preS2抗体。结论该纯化方法实用有效,纯化后的preS2(epi)S蛋白比S蛋白具有更好的免疫原性。Objective To purify the hepatitis B virus （HBV） preS2 （epi） S protein expressed in Pichia pastoris and study its immune effect. Methods Induce recombinant Pichia pastoris containing preS2 （epi） S gene with methanol, and purify the expressed product by sucrose zonal centrifugation, Butyl-S-Sepharose 4FF hydrophobic and DEAE-Sepharose Fast Flow ion exchange chromatography and identify by SDS-PAGE and Western blot. Prepare vaccine with the purified protein and perform potency test in mice. Determine the serum antibody concentration of mice by RIA and calculate ED50 and relative potency. Test the antibody against preS2 （epi） S by ELISA. Results The purified preS2 （epi） S protein with a relative molecular mass of about 27 000 reacted with specific antibody. The ED50 of 2 batches of vaccine prepared with the purified protein were 0. 35 and 0. 48μg/ml respectively,while that of reference vaccine was 0. 52μg/ml. The mean serum antibody concentrations induced by the 2 batches of vaccine were 808 and 784 mIU/ml respectively, while that by reference vaccine was 312 mIU/ml. The relative potencies of the 2 batches of vaccine were 1.5 and 1.1 respectively. Both the 2 batches of vaccine induced antibody against preS2 in immunized mice. Conclusion The purification method was practical and effective, and the purified preS2 （epi） S protein showed good immunogenicity compared with S protein.

关 键 词：乙型肝炎病毒 PRES2蛋白 S蛋白 纯化 免疫效果 

分 类 号：R373.21[医药卫生—病原生物学] Q786[医药卫生—基础医学]