Establishment And Application Of Indirect ELISA Method For Detection Of Mycoplasma Capricolum Subsp.Capripneumoniae Antibodies Based On 0507 Protein

Mycoplasma capricolum subsp.Capripneumoniae（Mccp）is the pathogen of Contagious caprine pleuropneumonia（CCPP）.The disease has a wide spread in China and has seriously impacted the development of China’s livestock industry.Timely monitoring of the epidemic situation of Contagious caprine pleuropneumonia is crucial for the health of goat and the healthy development of goat farming.Due to the current lack of serological detection technology for CCPP,we established an indirect ELISA method for the detection of antibodies against Mycoplasma capricolum subsp.Capripneumoniae by gene cloning,sequence analysis,and prokaryotic expression of the 0507 gene of Mycoplasma capricolum subsp.Capripneumoniae,and preliminarily tested 282 goat serum samples collected clinically.The results are as follows:1.Successfully cloned the 0507 gene of Mccp Shaanxi epidemic strain and analyzed its nucleotide sequence and encoded amino acid sequence.The gene has more than 99%homology with 13 reference strains,and is the closest genetic distance to the Chinese strain M1601,with no significant genetic difference from other reference strains.Amino acid sequence analysis showed that the gene had no signal peptide,no transmembrane region,27phosphorylation sites,multiple antigenic epitopes,and good hydrophilicity.Protein secondary structure prediction shows that there are 159 Alpha helix,44 extended strand,14beta turn and 90 random coil.2.The prokaryotic expression vector p ET-28a-0507 was successfully constructed.Determine the optimal expression conditions:Induce for 4 hours with IPTG at a final concentration of 0.4 mmol/L at 37℃.The protein was determined to be expressed in the form of an inclusion body.The induced protein was purified using His nickel column and refolded by urea dialysis to obtain 0507 protein with a protein size of 39 ku.After Western blot identification,the recombinant 0507 protein has a good reactivity.3.An indirect ELISA method based on 0507 protein for the detection of antibodies against Mycoplasma capricolum subsp.Capripneumoniae was successfully established.The best test condition for this detection method is to use 3μg/m L to 96-well plate was coated with 0507 protein and sealed with 5%skimmed milk powder for 60 minutes.The first anti goat serum was diluted 1:100 for 120 minutes.The HRP labeled rabbit anti goat Ig G was diluted 1:2500 for 60 minutes.The TMB was used for color development for 20 minutes.Finally,the critical OD₄₅₀value of the detection method was determined to be 0.221.The method has good specificity,sensitivity,and repeatability.The detection method established in this experiment was used to detect 282 goat serum samples collected clinically,of which37 were positive,with a positive rate of 13.12%.In summary,this experiment cloned and sequenced the 0507 gene of Mycoplasma capricolum subsp.Capripneumoniae;Secondly,a prokaryotic expression recombinant plasmid was constructed,and an indirect ELISA method for the detection of antibodies against Mycoplasma capricolum subsp.Capripneumoniae using 0507 protein as the coating antigen was established,provide technical support for the seroepidemiological investigation of Contagious caprine pleuropneumonia.