Prokaryotic Expression And Identification Of The Biochemical Activity Of Mycoplasma Pneumoniae Uncoupling Enzymes RuvA And RuvB In Pigs

Mycoplasma hyopneumoniae（Mhp）is a chronic pneumonia caused by Mycoplasma hyopneumoniae（Mhp）,also known as endemic pneumonia in pigs.Mhp infection is highly prevalent worldwide and causes significant economic losses to the pig industry due to increased costs of treatment and vaccination,reduced pig quality and increased mortality due to secondary infections.Pneumoniae and other bacteria such as Bordetella bronchiseptica and Haemophilus parvum to induce porcine respiratory syndrome.The current main methods of treating and preventing mycoplasma diseases,including Mycoplasma pneumoniae in pigs,are the use of antibiotics and vaccines.These methods can mitigate the harmful effects of the disease and reduce morbidity,but mycoplasmas are still detectable in the host after treatment and can easily lead to recurrence and persistent infection.Therefore,the exploration of effective drug targets and the development of anti-Mhp drugs are important for the control of Mycoplasma pneumonia in pigs,and may also provide lessons for the development of other anti-Mycoplasma drugs.It is generally accepted that the most common strategy for pathogenic microorganisms to evade host immune detection and control is antigenic variation.Homologous recombination between specific repetitive DNA elements（VNTRs）distributed in the mycoplasma genome is one of the most important ways of antigenic variation.The decapping enzyme RuvAB is an important class of enzymes involved in homologous recombination,but the mechanism involved in mycoplasma is unclear.Therefore,elucidation of the biochemical activity of RuvAB will provide a basis for further in-depth exploration of the molecular mechanisms involved in the recombination repair of mycoplasma,which in turn will assist in drug discovery using them as targets.In this study,we firstly identified the relationship between RuvAB and antigenic variation by q PCR and general PCR,and initially determined that RuvA and RuvB might affect the antigenic variation of Mycoplasma pneumoniae.Then the RuvAB protein was induced to be expressed by the prokaryotic expression system,and polyclonal antibodies were prepared using New Zealand rabbits to provide biological materials and experimental basis for subsequent experiments.Finally,different DNA template substrates were designed,and the substrate specificity and DNA binding ability of the two were analysed by gel migration assay（EMSA）,and the substrate affinity of RuvAB was resolved by surface plasmon resonance（SPR）.The details of this study are as follows:1.The homology of RuvA_Mhp,RuvB_Mhp and several other pathogenic mycoplasmas was identified based on protein multiple sequence comparisons,which revealed that RuvA_Mhp and RuvB_Mhp are highly conserved,suggesting that they may affect antigenic variation in M.hyopneumoniae like other reported mycoplasmas.Correlations between antigenic gene sequence variation and differences in transcript levels of RuvA and RuvB in M.hyopneumoniae were analysed between and within strains,respectively.The results showed that the transcript levels of RuvA and RuvB were higher in isolates with more repetitions of VNTRs;M.hyopneumoniae treated with DNA damaging agents showed length variation in the VNTR of the antigenic gene H4,and the transcript levels of RuvA and RuvB were up-regulated.The above results suggest that the sequence variation of Mycoplasma hyopneumoniae antigen is correlated with RuvAB expression.2.pET21a-RuvA and pGEX-4T-1-RuvB recombinant plasmids were constructed by ligating the target protein gene with a double-encoding plasmid.The RuvAB protein was induced with the help of E.coli HB101 strain and purified by using nickel affinity chromatography residence and glutathione affinity chromatography column to obtain high purity target protein.3.A polyclonal antibody to RuvA was prepared using New Zealand rabbits,and the potency and immunogenicity of the antibody were detected by ELISA and Western blot techniques.We obtained RuvA polyclonal antibodies with potency above 1:156,000 and high specificity.4.The DNA binding activity of RuvA_Mhp and RuvB_Mhp was analysed by surface plasmon resonance（SPR）combined with gel migration assay（EMSA）,and it was found that RuvA_Mhp could bind to single-stranded and double-stranded DNA substrates to form complexes;RuvA_Mhp was more capable of binding to double-stranded DNA substrates containing single-stranded tails（replication fork mimic structures）,and the 3’-overhang had a higher binding capacity than5’-overhang;the longer the substrate strand,the stronger the binding capacity of RuvA_Mhp;the addition of Mg²⁺had an inhibitory effect on the binding capacity of RuvA_Mhp;and RuvA_Mhp had a recruitment effect on RuvB_Mhp.However,RuvB_Mhp did not have a recruiting effect on RuvA_Mhp;RuvA_Mhp binds to ss DNA in the form of an octamer.In summary,this study focused on RuvAB_Mhp protein,identified the correlation between RuvAB_Mhp protein and antigenic variation,reported for the first time the process of plasmid construction,expression and purification of RuvAB_Mhp protein,as well as the preparation of rabbit multiple antibodies for RuvA_Mhp protein,and assayed and analyzed the binding activity of RuvAB_Mhpprotein to DNA substrate under in vitro conditions.It laid the foundation for the subsequent exploration of the molecular mechanism of RuvAB_Mhp-mediated homologous recombination mediating Mhp antigenic variation.