Construction And Characterization Of Wza-deficeint Mutant Of Haemophilus Parasuis

Haemophilus parasuis(HPs)is a conditional pathogenic bacterial of pigs,which can be caused severe systemic disease in appropriate condition,classically characterized by fibrinous polyserisitis,arthritis and meningitis.However,capsule is all the time speculated to be a significant virulence factor in HPs;and also Wza gene,which codes transport protein mainly responsible for capsular polysaccharide,has been found.But Wza of HPs gene still hasn't been well studied,no further information about biological founctions of itself and related genes with Wza has not been confirmed in detail.For the purpose of illustrating the roles of Wza in capsule formation and the virulence of capsule,this project is performed to construct Wza mutant of HPs;and by comparing difference of biological characterisitics including growth rate,mycelial morphology,phagocytosis assay,virulence test against mice,seroresistance test and antibiotic susceptibility,the ralations among role of Wza gene on capsule formation and virulence changing can be more understood.Then,the achievements are listed below:First,the homologous recombinant plasimid pUC18-LR-Kan is successfully constructed by Overlap PCR,using ZJ1208 strain and pET-28 as the gene template and pUC-18 as the verctor.Then,through natural transformation system,the pUC18-LR-Kan can be lead into cells of five screened HPs strains;At last,by identification of PCR and sequencing,the ZJ1208-?Wza mutant is successfully constructed.ZJ1208-?Wza mutant and ZJ1208 wild strain have no visual differences on colonial morphology.ZJ1208 wild strain has a faster growth rate of in vitro culture and a more viable count than ZJ1208-?Wza mutant;athough both can reach the maximum OD600 after 12 h culturing,ZJ1208 wild strain is just about 2.196,and wild strain1.246,at 8 h the logarithm of viable count about wild strain wild strain is 9.7634,mutant nearly 9.3222.Also the thinner capsular polysaccharide treated with Capsular staining method can be seen in both mutant and wild strain by microscopy,but the long-chain or long rod form in ZJ1208-?Wza can be formed by connecting with each other cultivated about 6h in vitro,whereas wild strain not.Moreover,ZJ1208-?Wza mutant has a faster ratio of sedimentation which can be seen in 2h,but the field strain has no this character.Antibiotic sensitivity test proves that the deletion of Wza makes mutant more sensitive to?-lactam than field strain.Phagocytosis test?serum resistance test and challenge test on mice further prove that the deficiency of Wza can weaken the ability of ZJ1208 to resist the phagocytosis by PAM and serum lethal effect,also decrease the aggressive and deadly characters in mice.