The Recombination And Expression Of R1 Repeat Region Of Mycoplasma Hyopneumoniae P97 Adhesin Gene With Escherichia Coli Heat-labile Enterotoxin B Subunit Gene And The Immunogenicity Assay Of Recombinant Protein

Swine mycoplasmal pneumonia(SMP),caused by Mycoplasma hyopneumoniae(Mhp),is one of the most important respiratory diseases in swine breeding.The infection rate is high,but the mortality is low.The disease would lead to significant economical losses in pig industry.The commonly used vaccines to control this disease are inactivated whole cells and attenuated live vaccine, whose bio-products costs are high but the efficiency are limited.The objective of this study was to develop a recombinant subunit vaccine rLTBR1 containing R1 region of P97 adhesin of M. hyopneumoniae and B subunit of the heat-labile enterotoxin of Escherichia coli(LTB).Inserted the sequence into the express vector pET-28a(+) and induced two recombinant proteins rLTBR1 and rR1.The protein rR1 was expressed in the form of solubility and the recombinant protein rLTBR1 was inclusion body.The recombinant protein was purified through affinity chromatography method.The purified rLTBR1 was also tested for its biological activity by studies on its ability to bind to ganglioside acceptor in ELISA experiments.The result indicates that rLTBR1 revealed GM1 ganglioside-binding capacity.The antigenicity was evaluated by the methods of SDS-PAGE and Western Blot.Both of the results were positive.The immunogenicity of rLTBR1 was detected also by BALB/c mice.The antibody level was evaluated through ELISA test.The results suggested that inoculated rLTBR1 through the intranasal(IN) or intramuscular(IM) route both produced specified anti-Mhp systemetic and mucosal antibody (sIgA).The group inoculated R1 by IM route also produced specified anti-Mhp systemetic antibody, but through the IN route didn't induce specified and mucosal antibody.Group administrated rLTBR1 through the IN route also induced IFN-γsecretion by lymphocytes.Howerver,the other groups didn't induced IFN-γ,compared to the negative group.In conclusion,LTB can enhance the mucosal and cellular immunity level through the IN route.Certainly,the immune responses of mice should not be extrapolated,so the potential of the rLTBR1 for the control of SMP requires further studies in pigs.This study suggested that rLTBR1 vaccine may establish a new strategy for preventing infection by Mycoplasma hyopneumoniae.