The Research On Clonling Of P30 And P40 Gene Of Mycoplasma Ovipneumoniae Y98 And Immunogenicity Of P30

Mycoplasma ovipneumoniae (MO) is a seriously harmful pathogen to sheep. In recent years, with the development of sheep industry, the economic loss was caused by the large range appearance of MO.In this study, Mycoplasma pneumoniae Y-98 was rejuvenation by Hayflick liquid medium, hyperimmune serum was obtained by immuning rabbit with all protein of MO as antigen, and the major outer membrane antigens of MO were identified by indirect ELISA and Western blot. The results showed that the protein of 66,40,36 and 30KD may be the major antigen of MO. The primers were designed according to the P30 and P46 gene of Mycoplasma hyopneumoniae(Mycoplasma Hyopneumoniae, Mhp),the P30 and P40 gene of MO were obtained via PCR.The sequences of P30 and P40 gene contained one TGA and four TGA respectively which coded Trp rather than the stop codon,and we employed a modified overlap extension-PCR method for site-directed mutagenesis of selected TGA codons. The result showed that the P30 gene sequence identity of MO with Mhp-P30 gene was 79%; the P40 gene sequence identity of MO with Mhp gene was 74%. The result of the predictions of the epitope and the transmembran showed that both of transmembrane and epitope of P30 amino acids were high identity,both of them were transmembrane protein. The membrane area,transmembrane region and the extracellular region were between 1-37,38-60 and 61-the last amino acid respectively. In summary,the P30 protein may be used as an antigen for the immunodiagnosis of MO.The P30 and P40 gene were constructed in the prokaryotic expression vector pET28a and the plasmids were transformed into the strain Rossta to obtain the recombinant strains which were named pET-TB30 and pET-TB40 respectively.The plasmids were transformed into the strain Rossta to obtain the recombinant strain Rossta(pET28a-P30) and Rossta (pET28a-P40).SDS-PAGE and Western blot were performed,and the result indicated one kind of 30KD protein was induced by IPTG from Rossta(pET28a-P30) and this protein was mainly in the form of inclusion bodies,but not the plasmid of pET-TB40.Animal protection experiments of engineered strains Rossta(pET-TB30) showed that the Serum immuned by 75ug and 1OOug protein respectively was Positive.The result of biopsy showed that confluent lesions were foramation by alveolus of the lung from the infectd mice,and the alveolus around lesions were thickening,swelling and degenerated.The vascular structure was damaged with bleeding.The arrangement of liver cells was not neat,and liver cells were empty.Kidney was gnlargement and capillaries around kidney were mild bleeding. Some epithelial cells of tubular were denaturation and the lacunar was bigger,while some mice immuned were normal in the tissues of lung liver and keny.Summary,the P30 heterologous proteins has a certain protective effect.