Prokaryotic Expression And Immunogenicity Assay Of Mycoplasma Hyopneumoniae P97

Porcine mycoplasma pneumonia is a chronic respiratory disease caused by Mycoplasma pneumoniae infection,which can cause cough and asthma in sick pigs under natural conditions.After the pathogen invades the healthy pigs,it causes the cilia to fall off or atrophy by adhering to the cilia on the surface of the upper respiratory tract,resulting in insufficient immune protection,which leads to other pathogens infection,which ultimately increases the difficulty of diagnosis and prevention of the disease.As there is no symptomatic drug for the disease at present,it can only be prevented and controlled by the existing vaccine.At the same time,mycoplasma culture conditions are harsh,can not be cultured artificially,so that the cost of vaccine production is high,and can only provide partial immune protection,so there is an urgent need to explore a new direction of vaccine research.In this study,a nucleotide sequence with high immunogenicity of 195bp was screened from the Mhp183 gene of Mycoplasma hyopneumoniae by using related information software technology,which was named Mhp183_195bp.Three Mhp183_195bpnucleotide sequences were linked from beginning to end to form a new nucleotide sequence,named Mhp183_615bp.After direct synthesis,Mhp183_615bp was cloned into p ET100 vector and expressed in E.coli.After purification,the expressed protein was successfully verified by SDS-PAGE and Western-blot.BALB/c mice were injected with purified protein after 0,7and 14 days of feeding.The mouse sera were collected on the 0th day and the 22nd day after immunization.The purified expressed protein was used as antigen,and the antibody level in mouse serum was detected by Western blot.At the same time,IL-2,TNF-?and IFN-?in serum of mice were detected by ELISA.Results:the protein with the size of 30k Da was successfully expressed,and the protein could react specifically to both His-Tag mouse monoclonal antibody and porcine anti-Mhp specific serum.22 days after BALB/c mice were immunized with the purified protein,the antibody of the expressed protein in the experimental group was positive,while that in the control group was negative,suggesting that the expressed recombinant protein had immunogenicity.The results showed that the expression of IFN-?at 22 days after immunization was 0.80,3.56,4.04,3.73 times higher than that at 0 days before immunization,and the expression of IL-2 at 22 days after immunization was 0.71,1.17,0.98 and 1.10 times higher than that at 0 days before immunization.After 22 days of immunization,the expression of TNF-?was 0.68 times as much as that of 0 days before immunization(control group),2.65 times(high dose group),2.60 times(middle dose group)and 3.72 times(low dose group).This suggests that the expressed purified protein has a good level of cellular immunity.The results of this study show that the expressed recombinant protein can be used as a candidate for a new type of Mhp vaccine.