Mining Specific Diagnostic Targets Of Mycoplasma Ovipneumoniae Based On Comparative Genomics

Mycoplasma ovipneumoniae(Mo)is one of the main pathogens causing Mycoplasma ovipneumoniae pneumonia in sheep and goats.The main clinical symptoms after infection are cough,wheezing,progressive emaciation and pulmonary interstitial proliferative inflammation.It is a worldwide infectious disease.The results showed that there were significant differences in RFLP profiles among isolates from different sheep farms,and multiple Mo strains with different gene structures were isolated from the lungs of the same diseased sheep,which indicated that the gene structures of the strains were different.At present,there is no genome-wide differential alignment to analyze the protein coding region(CDS)sequences of different strains and screen new species-specific diagnostic targets.Objective:(1)The interspecific and intraspecific CDs sequences of Mo were screened by comparative genomics.(2)Some intraspecific protein coding genes were selected as potential molecular diagnostic targets,and the specificity of the target,sensitivity of the established method and adaptability of clinical detection were verified by ordinary PCR.(2)Intraspecific protein coding genes were selected as candidate antigen targets to analyze their antigenicity,construct expression vector,express and purify the target protein,which was preliminarily evaluated by WB test Antigenicity and specificity.Methods:(1)The 14 published Mo genome sequences were screened for the first time,and the sequences with high homology were obtained.Then,the sequences were obtained with Mycoplasma arginine(Marg),Mycoplasma mycoides subsp.Capri(Mmc),Mycoplasma capricolum subsp.Capripneumoniae(Mccp),The specific coding sequence was obtained after comparing the genome of Mycoplasma in sheep respiratory tract,and then compared with the genome sequencing data of 51 local isolates to obtain Mo specific protein coding genes.(2)The optimal Mo specific protein encoding gene was used to find the conserved fragment shared with 51 Mo strains.Using primer on NCBI Blast program designed primers,selected the target gene with high G/C content,established and amplified DNA of 53 Mo strains in the nucleic acid sample plate,15 other mycoplasma and 18 ruminant bacteria in the nucleic acid sample plate,and tested 27 samples of the temporary bed samples to evaluate their specificity,and applied the most widely LMF1/LMR1 primers and their reaction systems(PCR Methods)to compare the optimal sequence as the target of Mo nucleic acid diagnosis.(3)The potential specific coding protein was selected by software evaluation.Combined with the prediction results of sequence structure function,the sequence meeting the requirements was constructed by p ET-30 a,and the recombinant protein was obtained by prokaryotic expression system expression,IPTG induced expression,Ni-IDA+ affinity chromatography column purification,and then the reactivity of recombinant protein was evaluated by WB.The recombinant protein was evaluated as Whether it can be used as a new target of serological diagnosis of Mo.Result:(1)The 14 Mo genome blasts of NCBI excluded the sequences with high homology with Marg,Mmc and Mccp.Finally,24 Mo interspecific specific candidate sequences were obtained.The 24 candidate sequences were re sequenced with the whole genome of 51 native Mo strains for blast.Finally,15 mo intraspecific candidate protein coding genes and 13 Mo intraspecific conservative and specific protein coding gene fragments were obtained.(2)Eleven pairs of primers were designed based on the conserved fragments of 15 candidate protein coding genes in 51 Mo strains.Finally,a pair of primers 7282 F / 2R was successfully designed,which could amplify a 288 bp target band from 53 strains of Mo DNA from different sources.However,when reverse disc DNA was used as template,the target band could not be amplified.The specificity of 728 2F / 2R primer was better than that of LMF1 / LMR1 primer.(3)The recombinant proteins 258 and 728 were successfully expressed and purified.WB analysis showed that 258 protein had certain reactivity and specificity,while 728 protein had certain reactivity,but its specificity needs to be further evaluated.Conclusion:(1)The 13 conserved and specific candidate protein coding gene fragments were obtained.(2)728 sequence can be used as a new molecular diagnostic target for Mo,and a new pair of Mo specific primers 728 2F / 2R was successfully designed.(3)258 recombinant protein has certain reactivity and specificity,and has potential as a new serological diagnostic target for Mo,but its application conditions need to be optimized.