幽门螺杆菌液体培养的研究

Study on liquid culture of Helicobacter pylori

目的 :建立一种适用于幽门螺杆菌 (Hp)大量扩增并提取致空泡变性细胞毒素 (Vac A)的液体培养方法。 方法 :采用TSB及 MHB培养液 ,加入小牛血清 (FBS)或环糊精 (CD)作为支持剂 ,比较其对 Hp的扩增效果及上清中空泡毒素活性的差异。结果 :TSB+CD组 Hp平均扩增速度及菌体湿重与 TSB+FBS组相比无显著差异 ,二者均显著高于 MHB培养液组 (P<0 .0 1)。 TSB+CD组上清蛋白浓度为 (2 .4 4± 0 .39) m g/ m l,显著高于 MHB+CD组。 TSB培养液组浓缩上清具有明显的空泡毒性 ,且蛋白电泳可见相对分子质量为 95 0 0 0的 Vac A,而 MHB培养液组无空泡毒性。 结论 :以 TSB为培养液 ,加入适量的CD作为支持剂 ,在不损失 Vac A活性的前提下 ,可明显增加浓缩上清液中的蛋白纯度 ,是大量提取和制备 Hp Vac A蛋白理想的液体培养方法。

Objective:To establish a method of liquid culture for extracting the VacA toxin of H.pylori . Methods:Tryptic soybroth(TSB), Mueller Hinton broth(MHB), and 2 kinds of support reagents:fetal bovine serum (FBS) and β cyclodextrin(CD) were used as liquid culture. The growth rate of H.pylori and vacuolating toxin activity in supernatants were observed. Results:There was no significant difference between TSB+CD and TSB+FBS group in density of H.pylori . The results of these 2 groups were significantly higher than that of culture containing MHB ( P <0.01). Protein concentration of TSB+CD supernatant reached (2.44±0.39) mg/ml,which was higher than that of MHB+CD group. SDS PAGE showed that the line of 95 000 (VacA) in the mixture of groups containing TSB had the vacuolating activity, which was not seen in the extract of MHB supernatants. Conclusion:TSB broth with CD in liquid culture of H.pylori can increase protein purity without the loss of VacA activity.