Isolation And Identification Of SY Strain Of Mycoplasma Ovipneumoniae And Gene Cloning And Analysis Of Hsp70 Protein

Mycoplasma ovipneumoniae is a highly contagious respiratory infectious disease of sheep.M.ovipneumoniae causes atypical pneumonia and can also work with many other bacteria to induce severe pulmonary infections.The lung tissue sample was collected from a sheep with obvious respiratory symptoms in a farm of Yulin,Shanxi province.The suspicious colonies were purified and identified by biological methods,and the bioinformatics analysis of HSP70 protein was performed.The lung tissues from the sick sheep were treated with PPLO broth medium,and purified with TSB-1 agar medium.The suspicious colonies were chosen and identified as M.ovpneumoniae SYS-1 by morphology,physio-biochemical assays and 16S r RNA sequence analysis.PCR with 16S r RNA specific primers amplified a361 bp target gene fragment,and BLAST analysis after gene sequencing revealed97.81%identity with the reference strain of Mycoplasma ovipneumoniae Y98(NCBI reference sequence:NR＿025989.1).SY-1 was selected and passed according to the bacterial liquid and liquid medium at 1:10.The growth curve revealed that the maximum amount of bacteria could reach 10~9CCU.The bacteria liquid at logarithmic growing period was selected,Antibiotic paper diffusion method was used.The results showed that SY-1 was highly sensitive to doxycycline,moderately sensitive to ciprofloxacin,and showed low sensitivity to ceftriaxone,gentamicin,benoxacillin,enoxloxacin and azithromycin.The genomic DNA of the SY-1 strain was taken as a template and with the standard PCR method,the HSP70 gene was successfully cloned,and gene sequencing and BLAST alignment analysis revealed 98.11%identity with the reference strain Y98.The predicted values of HSP70 protein transmembrane region were analyzed with the software TMHMM 2.0,Signal P V5.0 program was used for analysis of signal peptide cleavage sites,glycosylation sites were analyzed by Net NGlyc online software,and the tertiary structure was predicted by Alpha Fold online software.The results showed that the polypeptide chain of HSP70 had no transmembrane region and no signal peptide cleavage site.HSP70 contained 85phosphorylation sites and 35 serine phosphorylation sites.Through the predication of the secondary structure of the HSP70,it was found that the spiral,irregular coil,transfer,and extension chain were 48.43%,24.63%,7.44%and 19.50%.From the tertiary structure,it was found that HSP70 were mainly helical,It was basically consistent with the results of the reference sequence analysis.Through the isolation and identification of pneumonia mycoplasma SY-1 in sheep,this test provided clinical reference data for the effective prevention and treatment of M.ovipneumoniae in sheep in Yulin City,and laid a foundation for the laboratory diagnosis of the bacteria and the new vaccine research.