Prokaryotic Expression And Immunogenicity Research Of Multi-epitope Fusion Protein Of Mycoplasma Hyopneumoniae

Mycoplasma pneumonia of swine is a chronic respiratory disease,which is considered to be one of the main reasons for the growth and development of pigs,causing serious economic losses to pig industry.At present,pig farms mainly use attenuated vaccines or inactivated vaccines to prevent the disease.However,it is difficult to culture Mycoplasma hyopneumoniae in vitro,the production cost of the vaccine is high,and the protection is limited.Therefore,there is an urgent need to explore a new vaccine with good immunity.In this study,three highly immunogenic antigen fragments were selected from the Mycoplasma hyopneumoniae 232 strain through bioinformatics technology,and each fragment was repeated three times and then connected end to end with a flexible connecting peptide to form a multi-epitope named Mhp232_1092bpfusion gene is expressed in the Escherichia coli system.The expression product was identified by SDS-PAGE,and its expression form and optimal expression conditions were explored,and then the protein was expressed and purified in large quantities.The immunological activity of the purified protein was verified by Western-blot using mouse His-tag antibody and pig Mhp specific serum.The immunologically active fusion protein was emulsified with Freund adjuvant,and BALB/c mice were injected intraperitoneally on days 1,8,and 15.Collect the mouse sera before and on the 22nd day after immunization to detect the levels of IFN-γ,IL-2 and TNF-αin the serum to evaluate the level of cellular immunity;Western-blot test was performed using immunized mouse serum,and an indirect ELISA method was established to detect mouse-specific Ig G antibodies to assess the level of humoral immunity.The results showed that the Mhp232_1092bpfusion protein is a soluble protein with a size of about 60k Da and has the highest expression under the conditions of a final concentration of IPTG of 0.8 m M and induction at 15°C for 20 h.The serum IFN-γcontent of mice after22 days of immune fusion protein was significantly increased compared with that before immunization,increasing by 423.16 pg/m L（high-dose group）,467.74 pg/m L（medium-dose group）,and 269.52 pg/m L,respectively（Low dose group）;The content of IL-2 content increased by 145.16 pg/ml（high-dose group）,115.97 pg/ml（medium-dose group）,and 14.03pg/ml（low-dose group）;There was no significant difference in TNF-αcontent compared with that before immunization,with an increase of 10.04 pg/m L（high-dose group）,6.86pg/m L（medium-dose group）,and 12.37 pg/m L（low-dose group）,respectively.After immunization,the Western-blot test of mouse serum showed a positive reaction.The established indirect ELISA method was used to detect the mouse serum,and it was found that the mice in each immunization group produced high levels of specific Ig G antibodies.The research results show that the vaccine using Mhp232_1092bpmulti-epitope fusion protein as the immunogen has good effects in inducing mouse cellular immunity and humoral immunity,laying a foundation for the exploration of Mhp genetically engineered multi-epitope vaccine.