Cloning, Identification And Location Of Trichomonas Gallinae Adhesion AP65

Trichomonas gallinae(T. gallinae) is the aetiological agent of a common avian parasitic disease—trichomonosis. Trichomonosis could occur to pigeons with different species and ages and it produce a most fatal to nestling pigeons.The parasite infects the upper digestive tract of pigeons. Esophageal and respiratory obstruction leads to emaciation, starvation, or asphyxiation of pigeons. Trichomonosis has been reported as a global epidemic disease of pigeons and doves with a high prevalence. The drug treatment has many side effects, and could produce drug resistance strains. At present, it is necessary to find out an effective therapy in treating trichomonosis. Pathogenic mechanisms related to molecular studies will be an important future research directions. For mucosal microbial pathogens, it is crucial to bind to host cells. Physical damage and invasion would happened to host after adhesion to host cells. AP65 is a kind of important trichomonad virulence genes and shows a significant role among adhesion proteins. Adherence would be an important properties and AP65 would be a crucial adhesion protein for the mucosal parasite, T. gallinae. An exploration of the potential surface virulence factors of T. gallinae may lay a molecular basis in the pathogenesis and genetically engineered vaccine to control trichomonosis.This experiment includes 4 parts:1. Cloning and sequencing of T. gallinae AP65According to the known AP65 of T. vaginalis, partial segment of T. gallinae AP65 was cloned. Combing with 5’ RACE PCR and 3’ RACE PCR, two full-length sequences of T.gallinae AP65 were amplified and sequenced, named TgAP65-1(1766bp) and TgAP65-2(1759bp). Two genes contain a 1704 bp ORF coding 567 AA, respectively. The sequences were submitted to GenBank(KJ721787、KJ721788).2. Sequence analysis of T. gallinae AP65There are 94.22 % nucleic acid sequence homology and 95.6% AA homology between the two genes. The sequences were distinct with 5’sequenc. Homology analysis was performed among T. gallinae AP65 and T. vaginalis AP65 and 90.0% homology was calculated. T. gallinae AP65 showed 80.0% homology with hydrogenosomal malic enzyme of T. vaginalis. T. gallinae TgAP65-1 and TgAP65-2 respectively encoded aprotein(63057.81Da、63042.74Da) with pI8.15、8.48. A scan of database and the Comparative analysis with structure/function information of T. vaginalis AP65 revealed a short leader sequence, no GPI, several protein binding sites and two malic enzyme domains(88-278,280-533) in T. gallinae AP65. To address this situation, the prediction results of TgAP65-2 was used as an example.3. Polyclonal antibody preparation of T. gallinae TgAP65-2By the means of DNA recombinant, pPIC9.0k-TgAP65-2 vector was constructed. The recombinant plasmid was transfected into GS115 by LiCl. Methanol was used to induce and Ni-NTA was used to purify. A recombinant 65 KDa protein,as expected, was identified by Western-blot and immune BALB/c mice to preparation polyclonal antibody. Titer of polyclonal antibody was detected by indirect ELISA.4. Identification and location of AP65 expressed by T. gallinaeSDS-PAGE and Western-blot were performed to identify AP65 expressed by T. gallinae.The expression and location of T. gallinae AP65 were labeled by immunofluorescence. The result revealed that AP65 was located on the surface of T. gallinae, but the fluorescence is weak.