Identification And Analysis Of The In Vivo Induced Antigen Of Haemophilsu Parasuis SH0165

Haemophilus parasuis is one of the major pathogens that cause respiratory disease in pigs. Haemophilus parasuis infection, also known as Glasser's disease, is characterized by polyserositis and arthrithis, and meanwhile leads to meningitis and septicemia. Currently, all the studies concerning virulence genes of H. parasuis were carried out under the in vitro environment simulating natural infection and there is no definite virulent factors has been reported in detail.In vivo induced antigen technique is a high throughput immune screening method that does not require an animal model. Studying the pathogen genes specifically expressed in host by using sera of the host infected with target pathogen, the technique can easily and effectively identify the antigen genes of microbial pathogen expressed during an infection. It can be used in the identification of immunogenic antigens specifically expressed in vivo during acute and chronic infection, and it can also capture outer-membrane protein and secretive protein, and detect transient infection different stages and pathways of pathogen infection. It has been applied in variations of pathogens, especially human pathogens, and many significant virulence-associated factors were identified, laying theoretical foundation for the development of novel drug target and highly effective vaccines as well as the establishment of diagnostic methods.In this study, we targeted H. parasuis genes that interact with host and screened and identified in vivo expressed genes of H, parasuis isolate SH0165 through in vivo induced antigen technique. The genomic expression library of this strain was successfully constructed and swine recovery sera were prepared. The sera were attached by H. parasuis and E.coli to remove antibodies expressed in vitro. By multiple screening on the library with attachment sera,24 genes specifically expressed in vivo were obtained. Porcine immune sera with high potency of this strain were produced and in vivo expression of the identified genes was characterized by comparative identification between immune sera and infection sera. Our study includes following parts:1. Construction of H. parasuis genomic expression library:Select sequenced H. parasuis strain, SH0165 to extract genome DNA, digested into 0.5-3kb DNA fragment by restriction endonuclease Sau3AI. The fragments were cloned into prokaryotic expression vector pQE80L/81L/82L then tranformed into E.coli to construct genomic expression library. Containing over 80% recombinant plasmid, the library was verified by enzyme digestion and polymerase chains reaction, proved the insertion fragments'length was correct. The library was qulified to be applied to screening research.2. Preparation of serum antibody probe:Pigs were challenged with H. parasuis SH0165, collected the rehabilitation serum. The serum was absorbed with the precipitation and supernatant antigens of H. parasuis and E. coli lysis, repeated 4 times. Determining the result of absorbtion was tested by ELISA, the titer of rehabilitation serum decreased sharply and qualified to be used in library screening.3. Screening the in vivo induced antigens:We screened the IPTG induced expression library by western blot,39 positive clones were acquired after twice screening. These clones were sequenced and determined 80 ORFS in them. Positive clones including multiple ORFs were selected to be subcloned, each ORF was screened through Western blot, identified 24 in vivo expess genes.4. Confirmation of in vivo expressed genes:Prepared immune serum of H.parasuis SH0165. Screened 24 in vivo expressed genes by using E.coli absorbed immune and rehabilitation serum as primary antibody,75% of them reacted with immune rather than rehabilitation serum, proving these genes were expressed specificly in vivo.We chose gene gam and gene mreC which respectively encodes host-nucleasr inhibitor protein and rod shape-determining protein MreC/Cell shape-determining protein for prokaryotic expression and protein purification,through Western blot, the two in vivo expressed genes were identified on expression level.The H. parasuis genes screened out by in vivo induced antigen technique mainly belongs to the category of metabolism, regulation, adhesion, transport, cellular structure and antibiotic resistance. Whether some of them are the potential virulent factors of H. parasuis or some of them possess good immunogenicity needs further demonstration. Those genes can be applied as candidate genes for vaccines, and some genes sensitive in distinguishing immune sera and infection sera can be used as diagnostic antigens in the development of diagnostic methods.