PCR Diagnosis Of A Suspect Contagious Caprine Pleuropneumonia Case And Prokaryotic Expression Of 0297 Gene

Contagious caprine pleuropneumonia(CCPP)is an infectious disease caused by Mycoplasma capricolum subsp.Capripneumoniae(Mccp)that mainly ravages goats' respiratory system and highly contagious.CCPP is a serious hazard to goat farming in Africa and Asia,and is listed as a statutory report infectious disease by OIE.Mccp can infect goats of any age and sex,especially lambs.The main clinical symptoms are high fever,cough,purulent nose fluid.The main pathological changes are fibrinous inflammation in pleura and lungs.The morbidity rate can reach 80-90%,sometimes up to 100%,and the mortality rate is 60%-80%.At present,the isolation and identification of the pathogen is the primary evidence for Mccp diagnosis,but the nutrition requirements are harsh,so the isolation and culture are difficult.CCPP was first discovered in Algeria,1873,and spread to China in 1920 s,followed by widespread epidemics in Inner Mongolia,Ningxia,Gansu and Tibet,causing great economic losses to the goat breeding industry in China.In order to diagnose and prevent Mccp more effectively,this study diagnosed a suspect Mccp case in a dairy goat farm in Shaanxi Province using PCR method and performed prokaryotic expression of Mccp 0297 gene.The results were listed as following:1.Lung tissue was collected from a suspect Mccp case in a dairy goat farm in Shaanxi Province.The forward and reverse primers of Mccp 0297 gene were designed using Primer 6.0.To diagnose Mccp,the DNA were extracted and 0297 gene fractions were amplified by PCR.After seqencing,recombinant positive plasmids containing 0297 gene were constructed.The other 9 registered Mccp 0297 gene sequences in GenBank were compared and analyzed using bioanalysis software,then the structural of the encoding protein was predicted.In this study,Mccp 0297 gene was cloned and the recombinant positive plasmid pET-28a-0297 was successfully constructed.The sequencing results showed that the gene was 717 bp,encoding 239 amino acid residues.The nucleotide sequence similarity between the cloned 0297 gene and other 0297 genes from 9 reference Mccp strains was between 99.7% and 100%.Phylogenetic tree showed that the Mccp from this case was on the same branch as the reference Mccp strains apart from NZ-cp017125.Protein structure prediction showed that the 0297 protein encoded by this sequence is highly hydrophilic and may have multiple epitopes.2.Both recombinant pET-28 a plasmid with 0297 gene and the empty pET-28 a plasmid were extracted and transformed into BL21 competent cells.the optimal expression conditions were selected with IPTG and western blot analysis was performed.The optimization results showed that the optimal induction time of 0297 protein was 4 h,and the induced concentration was 0.8 mmol/L.By analyzing the supernatant of bacterial suspension,the supernatant and the sediment of bacterial cell lysis,it showed that the recombinant 0297 protein was an inclusion body protein with a size of about 43 ku.Western blot analysis showed a specific band,suggesting the recombinant 0297 protein could specifically react with the Mccp-positive serum identified and stored in our laboratory.In summary,Mccp was identified as its 0297 gene was successfully amplified with PCR,and the 43 ku recombinant 0297 protein were also obtained with prokaryotic expression system.Western blot assay showed that 0297 protein can specifically bind to Mccp positive serum with a good reactionogenicity.This study has a positive significance for the rapid lab diagnosis of Mccp and the development of Mccp novel epitope vaccine,providing information for the study of molecular biological characteristics and a basis for the further development of Mccp ELISA kits.