Construction Of DsbA Gene Mutant And Functional Studies In Haemophilus Parasuis

Haemophilus parasuis(Haemophilus parasuis, HPS) is an opportunistic pathogen which colonizatized with respiratory tract in pigs.With the poor outside conditions and body’s own immune system declined, HPS can lead to serous arthritis,meningitis, with fibrinous exudation as the main pathological changes. But about parasuis major virulence factor and its pathogenesis is not yet clear. In HPS we find a dsbA gene who encodes disulfide-folding enzyme.And the enzyme play a important role in the proper folding and accurate positioning of many secreted proteins and outer membrane proteins.In order to research the roles of dsbA in phagocytosis process, we constructed the dsbA gene deletion mutants the this experiment to study its adhesion and resistance to phagocytes.1. Construction of recombinant plasmid pK18-D1K and PK18-D2EAmplifing the upstream and downstream homology arms of dsbA1and dsbA2gene with primers designed on SH0165genome and plus USS sequence at both ends of homology arms.Connected the homology arms and corresponding resistance cassette with overlapping PCR,then digested the recombinant fragment and pK18mobsacB vector with same restriction endonucleases.After connection with T4Ligase between the recombinant fragment and pK18mobsacB vector, transformed into E. coli DH5a. After identification,we obtained the recombinant plasmid pK18-D1K, PK18-D2E without any nucleotide mutation.2. Construction and Screening of deletion mutants of dsbA gene with natural transformationTransformed recombinant plasmid pK18-D1K, PK18-D2E into HPS with natural transformation, we got dsbAl deletion mutant containing kan resistance and dsbA2deletion mutant containing erm resistance. Then PK18-D2E transformed into dsbAl deletion mutant and got a double-deletion mutant with kan and erm resistance.3. Identification of dsbA gene deletion mutantsAmplification of16S rRNA, resistance cassette,the target gene with PCR, preliminarily verified the deletion mutants; Amplificate the wild strains and deletion strains with same primers,and sequence alignment results with Blast indicated that resistance cassette has replaced the dsbA gene;Amplificate to cDNA with internal primer of target gene and resistance cassette indicated that the resistance cassette can be expressed but the target gene can not. With the corresponding gene of the gene downstream of the internal primers of the cDNA for the amplification of gene deletion strains, the deletion mutant proved to give no effect of polar.4. Function studies of dsbA geneBy plotting the OD6oo-time curve between wild strain and deletion strains,we can know that there is no difference in growth of different strains. And the same OD600corresponding the same number of viable cells.At same time,after incubation of bacterias and cells,we detected the lactate dehydrogenase (LDH) activity in the culture supernatant with LDH Cytotoxicity Kit. Through the incubation with PK-15cells and RAW,we found that dsbAl gene acts an important role in cytotoxicity and the cytotoxicity are not reflected in the PK-15.The results of adhesion assay indicate that:both dsbAl deletion and dsbA2deletion showed adhesion declined to PK-15,and dsbA2gene plays a more important role in adhesion stronger than dsbAl gene.The next phagocytosis assays suggest that:both dsbAl deletion and dsbA2deletion showed resistance to phagocytosis declined, and the ability of survival in RAW is declined obviously after dsbA2gene deleted.Besides, we detects the content of total after obtaining dsbA1, dsbA2deletion strains and double-deletion strain. Through the detection of each gene deletion strains and wild-type strain virulence gene expression, we found that in dsbAl deletion strain and double-deletion strain wza gene were downregulated of23.2and22.7folds. After extraction of total bacterial sugar and SDS-PAGE electrophoresis detected, double-gene deletion strains of sugar content slightly more than other strains, but no significant difference between the rest of the strains.