副猪嗜血杆菌自然转化方法的建立及条件优化

摘要

自然转化可用于构建副猪嗜血杆菌(Haemophilus parasuis,HPS)的基因缺失株,对于研究其毒力因子和致病机制有重要的意义.本研究采用控制变量法,对副猪嗜血杆菌自然转化过程中的实验条件进行优化.分别比较了点样时细菌不同生长阶段、点样孵化不同时长、转化体系细菌不同浓度、外源环磷酸腺苷(cyclic adenosine monophosphate,cAMP)不同浓度、外源质粒不同浓度及转化后平板孵化不同时间等条件下的自然转化频率.结果表明,副猪嗜血杆菌的平板转化实验在细菌处于对数生长后期(OD600≈1.5～1.7)时点样孵化,孵化时长为13h,体系中外源质粒的浓度大于0.1 μg/μL,转化后平板孵育5h时,副猪嗜血杆菌自然转化以较高频率发生.本研究确定了副猪嗜血杆菌自然转化过程中的最佳条件,为副猪嗜血杆菌毒力因子及致病机制等基因功能的相关研究提供了参考.%Natural transformation can be used to construct deficient mutant of Haemophilus parasuis (HPS),and it has significance on the study of pathogenesis and virulent factors.The experimental conditions of natural transformation are optimized by control variable methods.This research was conducted to obtain the optimal conditions for natural transformation of HPS.The transformation frequency was compared under the differential conditions,which including the bacterial phases (OD600＝0.15,0.40,0.70,1.00,1.30,1.50,1.60,1.90,2.00),bacterial incubation time (T1＝7,9,11,13,15,17,19 h),bacterial concentration (1/100 OD600＝0.1,0.2,0.3,0.4,0.5,0.6,0.7),the concentration of exogenous cyclic adenosine monophosphate (cAMP) (terminal concentration G1＝0.0,0.1,0.2,0.4,0.8,1.0,2.0,4.0,8.0 mmol/L),plasmid (terminal concentration G2＝0.000,0.005,0.010,0.020,0.040,0.050,0.100,0.250,0.350 μg/μL),and the incubation time after transformed (T2＝0,1,2,3,4,5,6,7 h).The results indicated that the transformation on agar plates of HPS occurred in a high frequency when the spotting bacteria was in the late-log phage(OD600≈1.5～1.7),the incubation time was 13 h,the concentration of exogenous plasmid was more than 0.1 μg/μL,and the incubation time after transformed was 5 h.The optimal conditions of natural transformation are determined,which lay a foundation for the research of virulence factor and pathogenic mechanism associated with Haemophilus parasuis.