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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 HBsFab抗体的产业化生产及临床研究奠定了基础
OBJECTIVE To investigate the high-density fermentation of anti-HBsFab antibody-fermenting engineering strain GS1 1 5 / Fab and the purification and identification of fermentation products. Methods Fermentation engineering bacteria GS1 1 5 / Fab was fermented at a high temperature in a 5 L fermenter at a fermentation temperature of 28-30 ° C with a pH range of 50-55 and a dissolved oxygen range of 2 0%. The three fermentation started when the OD60 0 value reached 400 ~ 0.250 ~ 250.030 0 ~ 35 0, the concentration of methanol was 0 5% ~ 1% The fermentation was terminated after about 96h of induction. The fermentation supernatant was affinity purified and the purified product was identified by ELISA. Results When the OD60 0 was 30 0 ~ 35 0, the control of the fermentation process and the expression of the target protein were started, and the target protein was expressed at a concentration of 245 mg / L. The fermentation supernatant was purified by affinity chromatography to obtain a recombinant Fab antibody with a purity of 98%. The antibody has high affinity and specificity for HBsAg antigen by ELISA analysis. Conclusion Fab yeast engineering bacteria in 5L fermentor high-density fermentation success, for the anti-HBsFab antibody industrial production and clinical research laid the foundation