The Research On Adhesion And Resisting Phagocytosis Of Wza In Haemophilus Parasuis

Haemophilus parasuis is a species of opportunistic pathogen in swine upper respiratory tract. It is pathologically characterized by fibrinous polyserositis, arthritis and meningitis. Capsular polysaccharides play an important role in the pathogenesis. This paper studied the effects about adhesion and resist phagocytosis of capsular polysaccharide export protein in the Haemophilus parasuis. Provide a theoretical basis for the molecular mechanisms of colonization and spread in the Haemophilus parasuis. The main research results are as follow:1. Construct the wza mutant about capsular polysaccharide export protein in HPSWe amplified the upstream and downstream fragments of wza, and added the USS (ACCGCTTGT) sequences on the primer of5’in upstream and on the primer of3’in downstream, then amplified the kan by PCR, and connected the three fragments by overlapping PCR and connected them with the vector of pK18mobsacB,It was named pK18-wza-UKD, the recombinant plasmid has been transformed into strain FX0103by natural transformation, we have gotten the wza mutant through kan resistant plate and we have verified the wza mutant by PCR, RT-PCR, Southern Blot and Western Blot.2. Comparison the biological characteristics of wild strain FX0103and mutant strain FX0103-△wzaThe growth curve of wild strain FX0103and mutant stain FXO103-△wza have been drown in0-24h, The result showed that after missing wza has not affected the growth of both strains; We found the capsular of wza mutant was thinner than wild strain’s by transmission electron microscopy; Extraction the total polysaccharide and extracellular polysaccharide of FX0103and FXO103-△wza to detect the express, We found expression of total polysaccharide of wza mutant was higher than wild strain and the extracellular polysaccharide of FXO103-△wza was lower than wild strain. Then we conducted serotype experiment of FX0103and FX0103-△wza, The result showed that the capsular polysaccharide export protein of bacterial may affect the ability of serotype.3. Capsular polysaccharide export protein is related to adhesion in HPSWild type strain FX0103and FX0103-△wza mutant were cultured with porcine kidney epithelial cells (PK-15), The results showed that the adhesion capacity of FX0103-△dwza was stronger than wild strain. The adhesion capacity was strongest both wild type strain FX0103and FX0103-△wza mutant at90minutes. The reason is that the strain FX0103-△wza which destroied the capsular polysaccharide export protein may expose the binding sites of the host, so that more bacteria adhered to the cell surface.4. Capsular polysaccharide export protein is related to resist phagocytosis of HPS Wild strain FX0103and0X0103-△wza mutant were cultured with mouse macrophages (RAW), The results showed that the resistant phagocytic capacity of wild strain FX0103was stronger than FX0103-△wza mutant. Then compared the intracellular survival ability of wild strain FX0103and FX0103-△wza mutant showed that there are significant differences at different time points and the survival ratio of FX0103-△wza mutant was lower than wild strain.