Abstract：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 C1=0.0, 0.1, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 8.0 mmol/L), plasmid (terminal concentration C2=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.
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