重组毕赤酵母菌高密度表达人源抗-HBs Fab抗体的研究  

Expression of recombinant humanized anti-HBsAg Fab fragment in genetically engineered Pichia pastoris in high-density fermentation

作　　者：陈文吟[1] 饶桂荣[1] 粟宽源[1] 邓宁[2] 余宙耀[1] 

机构地区：[1]解放军第458医院 [2]中山大学生命科学学院

出　　处：《解放军医学杂志》2004年第9期810-812,共3页Medical Journal of Chinese People's Liberation Army

基　　金：广州市科技局重大攻关项目基金资助课题 (编号 99Z0 1 0 0 1 )

摘　　要：目的　探讨抗 HBsFab抗体发酵工程菌GS1 1 5 /Fab的高密度发酵及对发酵产物的纯化与活性鉴定方法。方法　采用补料分批培养方法 ,在 5L发酵罐中对发酵工程菌GS1 1 5 /Fab进行高密度发酵 ,发酵温度 2 8～ 30℃ ,pH值范围为 5 0～ 5 5 ,溶氧范围 2 0 %以上 ;3次发酵分别于OD60 0 值达到 4 0 0～4 5 0、2 0 0～ 2 5 0、30 0～35 0时开始诱导 ,甲醇诱导浓度为 0 5 %～1 % ,经 96h左右的诱导后终止发酵 ,对发酵上清进行亲和纯化 ,并通过ELISA法对纯化产物进行活性鉴定。结果　在OD60 0 为 30 0～ 35 0时开始诱导有利于发酵过程条件的控制和目的蛋白的表达 ,目的蛋白表达量为 2 4 5mg/L。发酵上清通过亲和层析纯化 ,获得纯度为 98%的重组Fab抗体 ,该抗体经ELISA分析具有较高的HBsAg抗原亲和力及特异性。结论　Fab酵母菌工程菌在 5L发酵罐高密度发酵成功 ,为抗Objective To study the expression in high density fermentation of anti HBsAg Fab fragment in Pichia pastoris , and the purification and activity detection of expressed target protein. Method The high density fermentation of genetically engineered Pichia pastoris was proceeded in a 5L bioreactor using fed batch fermentation. The fermentation temperature was set at 28-30℃,the pH was 5 0-5 5, and the DO was kept over 20%. When the absorbance (OD 600 ) of the broth reached 400-450 (the first time of fermentation), 200-250 (the second time), and 300-350 (the third time), the induced phase was initiated, and the methanol concentration was 0 5%-1%. The fermentation ended after 96h's induction, the target protein was purified by affinity chromatography, and its activity was assessed by ELISA. Results It showed that the optimum initial cell density during methanol induced phase should be 300-350, which was good for control of the fermentation process and the expression of recombinant Fab. At the end of the fed batch phase, a yield of about 245mg/l of Fab was reached, and 98% purity could be reached as demonstrated by affinity chromatography. The results of ELISA showed that the supernatant of fermentation and the purified recombinant Fab could bind to HBsAg specifically. Conclusion The success of high density fermentation lays a sound foundation of mass production and clinical applications of recombinant humanized anti HBsAg Fab fragment

关 键 词：抗-HBS FAB抗体 高密度发酵 毕赤酵母菌 亲和层析 

分 类 号：R512.62[医药卫生—内科学]