The Cloning And Expression Of Glycerol-3-phosphate Oxidase Of Mycoplasma Ovipneumoniae And The Purification Of It’s Recombinant Product

Mycoplasma ovipneumoniae is one of the major pathogens which can cause chronic respiratory diseases in sheep. It can induce high fever, breathing, cough, anemia, growth retardation, progressive weight loss, and interstitial lung hyperplasia. In recent years, this disease has become a worldwide disease,and caused some economic losses of the breeding industry, seriously affected the healthy development of the sheep breeding industry. MO can not only infect sheep but also infect goats.The studies on Mycoplasma pneumonia and M.mycoides subsp. mycoides SC showed that glycerol is the major carbon source of mycoplasma and its metabolic product H2O2is major pathogenic factor. But the understand of the metabolism of glycerol in MO is unclear so far. So in this study, glpO was amplified by using extracted MO standard strain Y98genome as a template, which was based on the determination of genomics and analysis of MO standard strain-Y98strain (ATCC29419). Then the sequence of glpO was optimized to adaptate the expression system of E.coli. At last the recombinant protein was expressed in E.coli. by pET28a-glpOM and purified using Ni chromatography column. The results are as follows:1. MO standard strain Y98was expanded culture, and the whole genome was collected. Detected by electrophoresis, the genome with high purity, we could proceed to the next experiment.2. Through the whole-genome sequencing of the Mycoplasma pneumoniae standard strain-the Y98strain, Primers were designed besed on the sequence of MO Y98, glpO was Kanlified by PCR. The target gene was inserted into the pMD18-T downstream of the T7promote, then sequencing the target gene. Target gene nucleotide sequence was optimized.3. To constructed the recombinant vector pET28a as expression vector, Prokaryotic expression of target genes for1131bp, the recombinant strain was induced by IPTG, The obtained product was subjected SDS-PAGE,44kD size fusion protein was got.4. The target protein was purified by Ni chromatography column, a purified target protein was obtained.The success of expression and purification of glpO laid a solid foundation for the function study of glpO.