Studies On Adhesive Characteristics And Mucosal Immunity Of The Protein Of Mycoplasma Gallisepticum Adhesin

Mycoplasma gallisepticum (MG) is the aetiologic agent of chronic respiratory disease in poultry, which can cause the dropping of laying, reducing of the hatching rate and so on. It has brought serious economic loss to the poultry business. At present the key measure of the disease prevention is inoculating vaccine, because the attenuated vaccine and the inactivated vaccine themselves have some disadvantages, the development of novel vaccines receives the attention. Many studies showed that the surface adhesion protein (pMGA) of MG could attach to the host cells and then play the role of disease. pMGA, a major protoplasmic membrane protein, has the haemagglutinin activity. In this study, the pMGA1.2 gene was expressed in E.coli. The fusion protein cholera toxin B subunit (rCTB) was used as a mucosal adjuvant mixed with the fusion protein pMGA1.2 (rpMGA1.2) to immune the animals. Sera, nasal and tracheal washes were collected to detect the specific antibody levels, immune protection rate were calculated after MG challenge. The major results are as follows:1. Expression of Mycoplsma gallisepticum adhesin (pMGA1.2) and cholera toxin B subunit gene (ctb)rpMGA1.2 or rctb successfully expressed in E. coli BL21 (DE3). The expressed products were consistent with the expected size (rpMGA1.2 92.0KDa, rCTB 38.0KDa), and the expressed rpMGA1.2 mainly existed in the form of soluble protein, while the rCTB was mainly in inclusion bodies. Results of Western blot and GM1-ELISA assay showed that the rpMGA1.2 or rCTB had better immunogenicity.2. The adhesion characteristics of protein of Mycoplsma gallisepticum adhesin (pMGA1.2)The chicken embryos tracheal ring can grow well in DMEM medium Containing 10% newborn calf serum, and it can be used to test. The test samples of scanning electrometry microscopy images, rpMGA1.2 concentration was found in 100μg of MG chicken tracheal cilia off the best inhibition. The results showed that rpMGA1.2 was associated with MG adsorption.3. Mucosal immunity of Protein of Mycoplsma gallisepticum adhesin (pMGA1.2)rpMGA1.2 mixed with rCTB was used to immunize chickens via nasal and eyes, the results of ELISA showed that high level specific IgG and low level sepicific sIgA were detected in sera after the immunization; sepicific sIgA level in nasal and trachea washes was significant difference between rCTB+rpMGA1.2 group and rpMGA1.2 group (P<0.05), there was no significant difference between the two groups of different dose rCTB+rpMGA1.2 (10μg,20μg), and sIgA level in nasal washes was higher than it in trachea washes. Results of challenge showed that the low dose group of rCTB + rpMGA1.2 (10μg) had better immune response, the immunity protection ratio can be reached 77%. It shows that rpMGA1.2 can stimulate immuninty response.