Expression Of P159?P216 Gene Fragments From Mycoplasma Hyopneumoniae And Research On Adhesin Activity

Mycoplasma hyopneumoniae (Mhp) is the causative agent of Swine Enzootic Pneumoniae, a disease which caused by Mhp adhering to porcine respiratory ciliated epithelial cells. Adhesins of Mhp play an important role in the adhering process. In this study, adhesins molecule P159?P216 antigenic protein fragments of Mhp were expressed using the prokaryotic expression system, then the adhesin activity of these two proteins were studied. The research is started from the following aspects:1 Accroding to the gene sequence of Mycoplasma hyopneumoniae(Mhp) PI59 reported in GenBank, primers were designed by the use of Primer 5.0. Gene was amplified by PCR, then the gene was inserted into expression vector pET-28a(+), constructed the recombinant plasmid pET-28a(+)/P159. After it was successfully identified, the fusion protein was induced and expressed in E. coli BL21(DE3). When induced with 1% IPTG after 5h, the fusion protein got the highest amount. The fusion protein was purified by Protino Ni-TED 2000 Packed Columns. Western-blot analysis proved that the fusion protein have satisfactory immunogenicity.2 Accroding to the gene sequence of Mycoplasma hyopneumoniae(Mhp) P216 reported in GenBank, primers were designed by the use of Primer 5.0. The site-directed mutagenesis was performed by overlap-extension PCR (SOE-PCR) method at three sites of Mycoplasma hyopneumoniae (Mhp) P216 gene. Three condons which encoding Trp were mutagenized from TGA to TGG. Then the gene was inserted into expression vector pET-32a(+), constructed the recombinant plasmid pET-32a(+)/P216. After it was successfully identified, the fusion protein was induced and expressed in E. coli BL21(DE3). Induced with 1% IPTG after 4h, the fusion protein got the highest amount. Beacause of the fusion protein were expressed in the form of inclusion bodies, first the protein were denaturated by 8M urea, then using the method of dialysis to refold it into the target protein with biological activity. The fusion protein are purifed by Protino Ni-TED 2000 Packed Columns.Western-blot analysis proved that the fusion protein have satisfactory immunogenicity.3 Cilia protein were collected from the trachea of healthy pigs and diluted to 10?g/mL to coat microtiter plates. With P159?P216 antigenic protein fragments, microtiter plate test were studied. The results demonstrated that P159?P216 antigen protein fragments can adhere to porcine respiratory epithelial cell cilia.4 Mycoplasma hyopneumoniae 232 and PK15 cells were incubated for 4h, after that, Mhp/P46 monoclonal antibody (1:1000 diluted) was added and incubated for 90min, and then goat anti-mouse IgG-FITC (1:100 diluted) was added and incubated for 90min. The results were observed by fluorescence microscopy, and it illustrated that Mycoplasma hyopneumoniae 232 strongly adheres to PK15 cells.5 Mycoplasma hyopneumoniae P159% P216 antigenic protein fragments and SJPL cells were incubated for 4h respectively, then Mycoplasma hyopneumoniae 232 and the SJPL cell were incubated for 4h, adding Mhp/P46 monoclonal antibody (1:1000 diluted) incubated 90min, then addding goat anti-mouse IgG-FITC (1:100 diluted) incubated 60min.Fluorescence intensity using flow cytometry showed that the two proteins can produce inhibition of Mycoplasma hyopneumoniae adhering with SJPL cells, which can show that these two proteins have adhesion activity.Mycoplasma hyopneumoniae PI59?P216 antigenic protein fragment were expressed in the prokaryotic expression, then the adhesin activity of the two proteins were studied by microtiter plate adhesin test and indirect immunofluorescence. Through these studies, Mycoplasma hyopneumoniae protein adhesin model was initially established and it would be helpful for understanding the mechanism of adhesion of Mycoplasma hyopneumoniae, developing detection methods and studying the genetic engineering vaccines.