Preliminary Study On Protein Mycoplasma Gallisepticum Adhesin And The Protein Involved In The Interaction With PMGA

Mycoplasma gallisepticum (MG) is a persistent, highly transmissible chickenpathogen, and also the etiologic agent of chronic respiratory disease in chickens.Infections of the organism can yield significant losses in performance and associatedeconomics to all sectors of the poultry industry. Research has indicated that attachment ofMG to specific target cells along the respiratory epithelium is required prior to initiationof the disease process. In this paper, on the basis of preliminary studies, the followingexperiments had been done: firstly, we obtained the optimal conditions for soluble fusionprotein of pMGA1.2 in E. Coli BL-21; the product expressed in supernatant was purifiedusing GST·Bind Resin and a plenty of soluble protein was obtained, the final preparationwas 96% after purification by SDS—PAGE analysis, and that showed well immunologiccompetency by Western-blot analysis, secondly, Indirect IMF method was used to testinteraction between pMGA1.2 and the tissue paraffin sections of trachea, heart, lung, liver,kidney, thymus, bursa of fabricius, spleen, proventriculus and the duodenum of SPF chickembryo; thirdly, Dot-ELISA was applied to detect interaction between the membraneprotein extracted from the same tissues and pMGA1.2. The main results are as follows:1.Culturing at 22℃, for 8 hours, with pH of 7.0 and 0.05 mmol/L of IPTGconcentration were the optimal conditions for soluble fusion protein of pMGA1.2 in E.Coli BL-21. Under these conditions, the amount fusion protein was obtained in E. ColiBL21 exceeded 18.5% and the soluble fusion protein of pMGA1.2 exceeded 17%in thesupernatant.2. Results of indirect immunofluorescence assay revealed specific binding sites inthe trachea, heart, lungs, liver, kidneys, thymus, bursa of Fabricius, proventriculus andthe duodenum with the exception of spleen of SPF chick embryo for pMGA1.2.3. The membrane protein, extracted from trachea, heart, lungs, liver, kidneys,thymus, bursa of fabricius, spleen, proventriculus and the duodenum of SPF chick embryointeracted with the pMGA1.2; analysis with Dot-ELISA for afor mentioned tissuesrevealed positive results except for the spleen.4. In membrane protein extracted from these tissues except spleen, we found thesame product with Molecular weight approximately 30kD by SDS-PAGE analysis, so itwas concluded it's the possible protein interacting with pMGA1.2.