Screening And Characterization Of Mycobacterium Avium Gene Related To Alveolar Surfactant Stress Tolerance

Objective:Mycobacterium avium is the most important conditional pathogen in non-tuberculosis mycobacteria,the clinical symptoms are so similar to tuberculosis,which make difficulties for its prevention and treatment.The infection of Mycobacterium avium often associated with patients suffered from AIDS.The main stresses faced during Mycobacterium avium infecting the lung involve oxidizing agents,low pH and surfactant.In order to illuminate the molecular mechanism of Mycobacterium avium how to resist to bad environment of lung,we construct its genomic DNA library and get the target clone by selection with SDS.Investigating the functions of anonymous genes will give clues to understand why Mycobacterium avium is able to survive in human body and find new candidates of pharmaceutical.Methods:We obtain Mycobacterium avium genomic DNA,digested with Sau3A I,plasmid pBluescript SK（+）under the controlling of the inducible T7promoter digested with BamH I,and transformed into E.coli DH5αcompetent cells.The Mycobacterium avium genomic library is screening was used to selected the recombinant clones in 0.5%SDS stress,in order to get the target clone tolerance to surfactant.We insolated an anonymous protein.Overexpression of this protein in E.coli cells afforded significantly increase in SDS tolerance.Sequence anglicizing found the encode this protein was partial MAV-4012 and MAV-4292.Design primers to amplify MAV-4012 and MAV-4292 by PCR.The recombinant vectors pGEX-4012 and pGEX-4292were constructed by MAV-4012 and MAV-4292 fragments with the expression vetor pGEX-4T-3.The over expression of pGEX-4012 and p GEX-4292 in E.coli were detected by SDS-PAGE.The function of the proteins were confirm through tolerance with SDS,H₂O₂,NaNO₂ and low pH by the method of plate culture count.Result:The genomic library of Mycobacterium avium was successfully constructed.The titer of the library was 1.18×10 ⁴ cfu/mL.Thirteen colonies were randomly selected for colony PCR analysis and the size of the inserted fragment was between 250bp and 750bp,which was satisfied with requirement of library establishment.Two SDS-resistant clones were screened from the genomic library of Mycobacterium avium with the biological activity level screening method.Sequence analysis showed that the two clones were MAV-4012 and MAV-4292.MAV-4012 and MAV-4292 were respectively ligated with the vector pGEX-4T-3 and transformed into E.coli.A recombinant strain that overexpresses MAV-4012 and MAV-4292 protein was successfully constructed.Under various pressure conditions in vitro,overexpression of MAV-4012 and MAV-4292 protein can enhance the survival of recombinant bacteria under surface active pressure SDS conditions,and enhance the survival of recombinant bacteria under low pH conditions.Overexpression of MAV-4292protein only enhanced the survival of the recombinant bacteria under the conditions of active oxygen ROS,while overexpression of MAV-4012 protein made the recombinant bacteria resistant to both active oxygen ROS and reactive nitrogen RNS.Through bioinformatic analysis of MAV-4012 encoded protein,it is likely to be a transmembrane protein and has a Geopilin domain.The sequence homology and the specificity of the protein to any other protein were found by comparing the sequence of the MAV-4292 protein were not high.Conclusion:Two genes resistant to alveolar surfactant,MAV-4012 and MAV-4292,were screened from the M.avium genomic library.From the predicted physicochemical properties,functional sites,and structural characteristics,the expressed protein may be a valuable target for the diagnosis,treatment,or prevention of avian mycobacterial disease.