The Discovery And Verification Of Nuclease Function For Secreted Protein RV1677of Mycobacterium Tuberculosis

Bovine tuberculosis is a chronic bacterial disease of animals and humans caused by Mycobacterium bovis.Mycobacterium bovis is a member of the M.tuberculosis complex?MTC?.In this MTC,every member has a identical 16s RNA sequences and over 99.9%identity of their genome sequences.There are a few research reports about non-restricted nuclease in the pathogens of Streptococcus and Staphylococcus aureus,but it has not been reported in the Mycobacterium tuberculosis complex.as a secreted protein of the Mycobacterium tuberculosis with thioredoxin function,RV1677 is still unknown in other biological functions.In this study,we demonstrated that RV1677 is a Mg²⁺-dependent nucleic acid enzymes.This finding may lay the foundation for the pathogenesis research of TB.In this study,we use mycobacterium tuberculosis standard strain H37Rv as the research object,and amplified this gene by PCR.RV1677 genes were connected to the prokaryotic expression vector pET-22b and eukaryotic expression vector pEGFP-N1.We use prokaryotic expression system to obtain the RV1677 protein and further purificated it.We prepared polyclonal anti-rabbit serum with the the RV1677 protein.RV1677protein was confirmed by mass spectrometry.Purified RV1677 proteins was used to a series of verification experiment of biochemical properties and enzymatic properties.At the same time,RV1677 gene with GFP tagged eukaryotic expression vector was transfected into Hela cells,and then laser scanning confocal microscope was used to detect it.The experimental results showed that the RV1677 protein owed a non-restricted exonuclease activity.We confirmed the predieted DNase activity of RV1677,which is metal-dependentand(Mg²⁺).The optimum reaction conditions are pH6.5 and 37?.The RV1677 protein has a strong heat-resistant activity which can play a role of Dnase as in the reaction conditions of 70?.After transfecting RV1677 gene in eukaryotic expression system,we found that it caused cell multi-polarization phenomenon and has the ability to degrade extracellular chromosome in Hela cells.These experimental results suggest that as a secreted Dnase of Mycobacterium tuberculosis the RV1677 protein may play an important role in the pathogenic process of M.tuberculosis infection.