Screening Of In Vivo Induced Antigen Of Haemophilus Parasuis And Construction Of Eukaryotic Expression Library

Haemophilus parasuis(Haemophilus parasuis,HPS) is a non-mobility and multi-form gram-negative bacilli bacteria,the bacteria belong to the Pasteuellaceae family. HPS is the pathogen of Glasser disease,it causes multiple serositis,arthritis and meningitis.Due to the high morbidity and mortality,the disease has caused tremendous economic losses to the hog industry around the world.At present,the research of Haemophilus parasuis is still at the initial stage,especially on the molecular level.This study was designed to identify the antigens expressed only in the host which might be used as the candidates of poetentional target for novel vaccine,drugs and diagnostic methods.we choose the dominant strain SH0165 prevalent in China.Firstly,we obtained the DNA fragments between 1kb and 5kb by digesting SH0165 genome with Sau3 A I.These fragments were then cloned in to the expression vector pET-28a,pET-28b,pET-28c. The recombinant plasmids were transformed into the E.coli BL21(DE3) to constract the prokaryotic expression library.The in vivo induced antigentechnique(IVIAT)was used to screen the library.Briefly,the gene expression of the bibrary was induced on LB agar plated with IPTG and blotted onto nitrocellulose membrane.The sera from naturally infected pigs were pooled and exhaustively absorbed respectively with bacterial body and the lysis of both E.coli BL 21 and HPS in vitro,thus leaving only in vivo expressed antibodies against HPS antigens.The absorbed antisera was probed the nitrocellulose membrane and the positive clones were developed by HRP coupled goat IgG to pig IgG and the substrat DAB.The inserted ORFs in the positive clones were sequenced with T7 promoter, and the sequence were then aligned with BLAST N on NCBI.As a result,the expression library of SH0165 strain SH0165 contained about 3.0×10~3 plasmids,which represented more than 80%of all the fragment which met with the theoretical requirements.After screening,sequence analysis and BLAST,we finally confirmed 6 ORF including glycerol ester phosphate kinase(phosphoglycerate kinase),3-oxygen acyl[acyl carrier protein] reductase(3-oxoacyl-[acyl-carrier-protein]synthaseⅠ),iron transfer protein(transferrin binding protein) and there conservative assumption protein genes.The further identification was being undergone.In addition,we constructed eukaryotic expresstin vectors by cloling the genome fragments into pcDNA3.1.