百日咳毒素、丝状血凝素和黏附素的纯化

目的采用新型层析介质对无细胞百日咳疫苗百日咳毒素(pertussis toxin,PT)、丝状血凝素(filamen-toushemagglutinin,FHA)和黏附素(pertactin,PRN)3组分进行分离纯化。方法调整PT和FHA发酵液上清的pH值和电导后,PT经Capto SP ImpRes和Capto MMC进行两步层析,FHA经Capto SP ImpRes进行一步层析;PRN热处理原液经Capto Adhere和Capto SP ImpRes进行两步层析。纯化产物经4%～12%NuPAGE进行鉴定,ImageQ分析纯度;LC-MASS(Thermo LTQ Velas)进行蛋白质的质谱检测;PT和FHA经BiaCore T200进行定量分析,并计算各步回收率。结果纯化后,无细胞百日咳疫苗3组分纯度均达95%以上;PT和FHA的总回收率均在30%左右;电泳图谱上的各组分条带经LC-MASS鉴定,均与Uniprot Bordetella pertussis数据库数据高度同源。结论采用新型层析介质有效纯化了无细胞百日咳疫苗PT、FHA和PRN,提高了抗原纯度,大大缩短了生产时间。

Purification of pertussis toxin, filamentous haemagglutinin and pertactin

Objective To isolate and purify the pertussis toxin(PT),filamentous haemagglutinin(FHA)and pertactin(PRN)of acellular pertussis vaccine by using a novel chromatography medium.Methods The pH value and conductivity of fermentation liquid of PT and FHA were adjusted,based on which PT was purified by Capto SP ImpRes and Capto MMC chromatography,while FHA by Capto SP ImpRes chromatography.The purified samples were identified by 4% ~ 12% NuPAGE and analyzed for purity by ImageQ and for protein mass spectrum by LC-MASS(Thermo LTQ Velas).PT and FHA were analyzed quantitatively by BiaCore T200,and calucated for recovery rates at various steps.Results The purities of PT,FHA and PRN reached more than 95%.The total recovery rates of PT and FHA were about 30%.LCMASS showed high homologies of bands of the three components on electrophoretic profile to those in databank.Conclusion The PT,FHA and PRN components of acellular pertusis vaccine were purified effectively by novel chromatography media,while the antigen purities increased,and the time for production was shortened remarkably.