Screening The Protein Of Actinobacillus Pleuropneumoniae Trimeric Autotransporter Adhesin In Lung Tissue

Trimeric autotransporter adhesin (Trimeric autotransporter adhesins, TAAs) are very important virulence protein for adhesion on outer membrane of many Gram-negative bacteria.lt was important in the process of bacterial adhesion, invasion ability, planting, film-forming, self-curing resist serum for bacteria. After adhesin by binding to the cell surface of the substrate or recept promoter intracellular signaling cascades triggered pathogen interaction with the body, and therefore bacterial adhesion in host pathogen interactions with the host immune system plays an important role.1. Construction and identification of bait yeast plasmid Adh geneAdh gene sequence to NCBI published based on primers designed from the APP 5b stored in our laboratory strain amplified Adh gene and its size is about 1600bp. Then build PGBKT7-Adh recombinant bait plasmid, the bait plasmid by PCR and sequencing showed that the inserted gene sequence Adh no frameshift mutation, indicating PGBKT7-Adh recombinant plasmids were successfully constructed.The PGBKT7-Adh recombinant plasmids were transformed into yeast AH 109 screened on SD/-Trp capable of growing yeast colonies for PCR identification, it showed that about 1600bp in PGBKT7-Adh recombinant plasmid was successfully transfected into yeast AH109, the Construction Adh protein-containing yeast two-hybrid bait plasmid. Bait plasmid was self-activation and toxicity of its own verification showed that non-toxic and non-self-activation phenomenon, indicating that the bait plasmid construct without interference, can be used for subsequent screening verification.2. Lung Library Quality AssuranceAfter lung tissue preserved E. coli and yeast library were plated store in our laboratory, respectively picked different single colony PCR library plasmid, the results show the library tape mostly distributed around 1000bp in line library fragment size requirements. Secondly, the lung capacity of E. coli was identified bacteria showed 5ml of total storage capacity of 0.9 x 107CFU. And then the lung tissue yeast library titer determination and defect growth media verification, defect culture showed capable SD/-Leu flat growth, not in the SD/-Trp/-Leu, SD/-Trp/-Leu/-Ade/-His flat growth. Titers showed greater than 2 X107 cfu/ml, showed a complete library. Taken together the results show that lung tissue can be used in a yeast two-hybrid library screening.3. Yeast two-hybrid screen positive interacting proteinThe bait plasmid laboratory preserved lung tissue cDNA library plasmids were transformed into yeast Y187 in. Growth and coloration principle according to deficiency of the yeast two-hybrid screening, after co-transfection were picked on SD /-His/-Ade/-Trp/-Leu/-X/-A large blue colonies grown by plasmid PCR and sequencing than for initial screening of interacting proteins. The results showed that after two rounds on SD/-His/-Ade/-Trp/-Leu/-X/-A pick 131arger blue yeast colonies grown, plasmid PCR and sequencing, the NCBI ratio analysis showed that four colonies having the possibility of interactions, initially identified four possible positive interactions with Adh protein, respectively zinc finger protein, containing I-platelet binding motif proteolytic polyprotein like metalloproteinases, HLA regulatory molecules-Ⅱ molecules, branched chain amino acid transaminase 1. Then analyze biological characteristics and functional sites interacting proteins and predict the potential function of Adh.This research is for further studing the function and structure of trimeric autotransporter adhesin (Adh) lay a foundation.