Screening And Identification Of Immunogenicity Proteins Mycoplasma Ovipneumoniae Strain IK3-3

Mycoplasma ovipneumoniae(MO)is the main cause of infectious pleural pneumonia in sheep.The disease is a highly infectious and high incidence chronic respiratory disease.In China,the pathogen has been isolated from sheep in many provinces.Cause huge economic losses.There are currently few commercial vaccines used to prevent the disease,and commonly used whole-bacterial inactivated vaccines have a shorter immune protection period.The subunit vaccine is the current research hotspot of Mycoplasma vaccine.This experiment screened the immunogenic protein of Mycoplasma ovipneumoniae virulent strain IK3-3 to provide ideas for the development of a new vaccine against Mycoplasma ovipneumoniae.In this study,based on the complete genome sequence determination of Mycoplasma ovipneumoniae IK3-3 strain,preliminary isolation and identification of MO-related immune proteins through proteomics technology,and further verification of its immune function through molecular biology techniques.First,a proteomics technology platform for MO whole bacterial proteins was established.The whole bacterial protein of Mycoplasma ovipneumoniae IK3-3 strain was successfully extracted.After treatment with 2D Clean-up Kit,the protein components were successfully separated using two-dimensional electrophoresis(2-DE),and highly repeatable.The stable electrophoresis pattern laid a good foundation for the subsequent research of MO proteomics.An average of 96 protein spots were detected in the repeating gel.The protein gel was transferred to Western-blot with Mycoplasma ovipneumoniae IK3-3 rehabilitation serum,and the two-dimensional electrophoresis pattern of MO IK3-3 strain and Western-blot pattern were performed.Compare and screen for immunogenic protein spots.The MALDI-TOF time-of-flight mass spectrometry was used to identify the highly immunogenic protein spots,and the amino acid sequence of the immune-related protein was obtained.The coding sequence of the corresponding protein was matched from the gene sequence of MO IK3-3 strain.All six protein spots were successfully identified,two of which were enzymes,which laid the foundation for the follow-up study of MO immune-related proteins.The optimized EF-Tu protein was codon optimized and whole gene synthesized,and the recombinant gene was inserted into the expression vector p Czn1.After induced expression by IPTG,a fusion protein with a molecular weight of approximately 45.4 k Da was obtained.High-purity protein of interest.Western-blot experiments were performed on the target protein with Mycoplasma ovipneumoniae high immunity serum and rehabilitation serum.The results showed that EF-Tu protein had a strong immune response with serum antibodies of Mycoplasma ovipneumoniae,and had good immunogenicity.Western-blot results of bacterial protein two-dimensional electrophoresis gel.Finally,an indirect ELISA detection method based on EF-Tu recombinant protein was established with good specificity and sensitivity.In the detection of clinical samples with the Mycoplasma ovipneumoniae indirect hemagglutination kit on the market,the compliance rate reached 77.5%,The research and prevention of Mycoplasma ovipneumonia in sheep has laid a foundation for further research.