Screening And Characterization Of Mycobacteriumaviumgene, Protects Ecoli From Nitrosative And Oxidative Stress

Mycobacterium avium-intracellular complex (MAC) is a conditional pathogen, the clinical symptoms are so similar to tuberculosis, which make difficulties for its prevention and treatment. Macrophage as a major host cell of MAC has so many bactericidal mechanisms, such as acids, ROS (reactive oxygen species), RNS (reactive nitrogen species), lysozymes, antimicrobial peptides, and so on. Theoretically, all of these are enough to kill pathogens. However, MAC can not only survive, but also multiply in macrophage. In order to illuminate the molecular mechanism of MAC how to resist to bad environment of macrophage, we construct its genomic DNA library and get the target clone by selection with NaNO2. All the work will lay the foundation for further research.Firstly, we obtain MAC genomic DNA by CTAB mean, digested with Sau3A I, plasmid pUC19 digested with BamH I, the connective products are transformed into JM109 competent cells, and construct genomic library. Secondly, the genomic library is screened with NaNO2 in order to get the target clone tolerance to RNS. Thirdly, analyze the insert gene sequence by bioinformatics. Fourthly, design a pair of primers to amplify MAC-noA and establish recombinant expression vector. Fifthly, use SDS-PAGE to detect the over expression of MAC-noA in Ecoli. Sixthly, confirm the function of MAC-noA through tolerance with NaNO2, GSNO, H2O2 and SDS by the method of plate culture count or the measure of absorbance value. Lastly, analyze physicochemical property and space conformation of the encoded protein through bioinformatics software and online software.As a result, we successfully constructed MAC genomic DNA library. The titer of library is 8×103cfu/ml. Insert fragment is 100-750bp, conforming to the requirement for a library. After tolerance test, we got a purposed gene (possessed 390bp) which had two reading frame, one was the target gene (naming noA), and another was part of MAC 104 p49 sequence, on two chains respectively. So, we researched a whole ORF (MAC-noA) next. Our research showed that, the survival ability of bacteria with MAC-noA was higher than the control group in test with NaNO2, GSNO and H2O2, but did not have survival advantage in SDS. Everything we do, find a new gene (MAC-noA) of MAC resisting NaNO2,GSNO and H2O2, this is a new gene resisting to both RNS and ROS. From analyzing physicochemical property, functional sites and space conformation, we can deduce that MAC-noA would add a possibility for macrophages to control MAC diseases through inhibiting MAC-noA. □...