培养条件对百日咳杆菌发酵中百日咳毒素及丝状血凝素表达的影响

Effect of culture condition on expressions of pertussis toxin and filamentous haemagglutinin in fermentation of Bacillus pertussis

目的探讨培养条件对百日咳杆菌发酵中百日咳毒素(pertussis toxin,PT)和丝状血凝素(filamentous haemagglutinin,FHA)表达的影响。方法采用300 L全自动发酵罐进行百日咳杆菌发酵培养,培养基为百日咳S-S培养基,搅拌通气培养,培养量200 L,温度3537℃,培养时间40 h,接种比例1∶121∶15。试验分为高PT表达组(搅拌转速为160 r/min,搅拌叶片为直叶,有挡板,培养020及2140 h的通气量分别为600和400 L/min)、高FHA表达组(搅拌转速为220 r/min,搅拌叶片为弯叶,无挡板,培养020及2140 h的通气量分别为400和600 L/min)及对照组(搅拌转速为180 r/min,搅拌叶片为直叶,有挡板,通气量为500 L/min),每组平行培养3批培养物。于培养结束时采用ELISA法检测PT及FHA含量。结果高PT表达组PT含量平均值达3.16μg/mL,比对照组(平均值2.23μg/m L)提升了41.7%(P<0.05);高FHA表达组FHA含量平均值达21.37μg/mL,比对照组(平均值11.00μg/mL)提升了94.3%(P<0.05)。结论百日咳发酵工艺中,高搅拌剪切力和较低的溶氧水平有利于PT的表达,低剪切力和高溶氧水平有利于提高FHA的产量。

Objective To investigate the effect of culture condition on expressions of pertussis toxin(PT)and filamentous haemagglutinin(FHA)in fermentation process of Bacillus pertussis.Methods B.pertussis was subjected to fermentation culture with S-S medium in fully automatic 300 L fermenter in ventilation.The volume,temperature,time and MOI for culture were 200 L,35~37℃,40 h and 1∶12~1∶15 respectively.The test was performed in three groups,three batches for each.The stirring speed in high PT expression group was 160 r/min,while straight mixing blade with baffle was adopted,and the ventilation volumes for culture for 0~20 and 21~40 h were 600 and 400 L/min respectively.In high FHA expression group,the stirring speed was 220 r/min,while curved mixing blade without baffle was adopted,and the ventilation volumes for culture for 0~20 and 21~40 h were 400 and 600 L/min respectively.However,in control group,the stirring speed was 180 r/min,while straight mixing blade with baffle was adopted,and the ventilation volume was 500 L/min.The PT and FHA contents were determined by ELISA after completion of culture.Results The mean expression level of PT in high PT expression group was 3.16μg/mL,which was 41.7% higher than that in the control group(2.23μg/mL)(P<0.05).However,the mean expression level of FHA in high FHA expression group was 21.37μg/m L,which was 94.3%higher than that in control group(11.00μg/mL)(P<0.05).Conclusion In the fermentation process of B.pertussis,high stirring shear force and low dissolved oxygen(DO)level were beneficial to the PT expression,while low stirring shear force and high DO level were beneficial to increase of yield of FHA.