Screening And Sequence Analysis Of Interaction Proteins Of Swine Lung Tissue With Functional Domain Of Apa1Autotransporter Adhesin

Trimeric autotransporter adhesin (TAA) is one of the most important virulence factors on thesurface of Gram-negative bacterium. It was important for bacterium in adhesion, invision,colonization, biofilm, auto-agglutination and serum resistance. Pathogen interacts with hostfollowing by adhesion induces intracellular signaling cascade though binding extracellular matrixproteins or receptors on the host cell suface, it is so that the interaction of bacteria adhesin withhost is the trigger point of immune response between pathogenic and host.In earlier research, we have found complete trimeric autotransporter adhesion named Apa1inActinobacillus pleuropneumoniae (APP) serotype5b strains L20. We have improved that the headdomain Adh (located at68~612aa) and the stalk domain BD3(located at2464~2574aa) play animportant role in adhesion, autoarrigation, biofilm formation and pathogenicity of APP byprokaryotic expression, knockout and comparison on animal model. Based on these results, weinmmued annimals by activated Adh and BD3proteins which was gained from prokaryoticexpression and purification in the purpose to get the polyclone antibody. We acquired theinteraction proterin by co-immunoprecipitation, following MALDI analysis and searching inprotein database, and identify the protein as keratin1. We constructed cDNA library of swine lungtissue and screened the sequence of keratin1by specific primer in the purpose to get deeperresearch about the relationship between Apa1with Keratin1. Based on the sequence we obtained,we predicted the characters though biological software and online prediction software. It showslow homology between the sequence no matter with keratins from swine or human, this mayrelated to the diversity between keratins and diversity distribution on different tissues. Based onthe analysis of amino acid, it shows that there was about1transmembrane domains consisted inthe protein, the main secondary structural elements were α helix and β fold, at the position of82~362aa existed a α subunit of G protein and515~552aa is a thrombin-sensitive protein type Irepeat domain. The α subunit united with β and γ subunit formed the heterotrimeric G proteinwhich forms the most common signal pathway system with G protein-coupled receptor. The thrombin-sensitive protein type I repeat domain is a multimeric multidomain glycoproteins whichis involved in complement pathway, cell-cell interaction, inhibition of angiogenesis and apoptosiss.These contents provide reliable references for the next research about protein interaction betweenTAAs and host cell receptor, the relationship between structure and function of TAA, andpathogenesis mediated by TAA.