The Effect Of M.smeg-2395 On Mycobacterail Growth By The Down-regulation Of Antisense RNA

Tuberculosis(TB),which is an infectious disease caused by Mycobacterium tuberculosis(Mtb),remains a major threat to global health.Nowadays,the number of TB cases and deaths remains still large due to MDR-TB(multi-drug-resistant,MDR),XDR-TB(extensively drug-resistant,XDR)and HIV-positive TB.There were an estimated 9.6 million new TB cases and a total of approximately 1.5 million TB deaths in 2014.The global burden of TB is enormous,especially,in Asia.The treatment of XDR-TB patients in particular remains very unsatisfactory and more effective regimens for this condition are urgently required.In recognition of the crucial role of research in fighting against Mtb pathogen and the requirement to reach reduction in TB incidence and mortality,many efforts to develop novel targets and their inhibitors have been implemented in the past decades.To combat with this pathogen of Mycobacterium tuberculosis,the D-Alanyl-DAlanine ligase(Ddl A)encoded by Rv2981 c,which is involved in peptidoglycan synthesis of cell wall in Mycobacteria,is chosen as an objective in this study.The essentiality of ddl A had been confirmed by mean of transposon mutagenesis which was implemented by Sesseti in 2003 in Mycobacterium tuberculosis.In addition,D-cycloserine,a second-line anti-TB drug at present,had been poven to taget on Tb-Ddl A,but its application to treat Tubeculosis was restricted due to the strong side effect.[D-cycloserine can activate NMDA(N-methyl-D-aspartic acid receptor)to cause the neurous system to be damaged.] Therefore,Ddl A had demonstrated as a potential anti-Tb drug target.In this study,we focus on the revealing of the function of Rv2981 c gene by over-expression of Ddl A and its inhibitory mechanism by the down-regulation of antisense RNA.The project herein was undertaken in two aspect:1.Identification of D-Alanyl-D-Alanine ligase activity.The sequence of Rv2981 c gene was obtained to construct the inducible expression vector of p Cold-Tb-ddl.Then,Ddl A protein was purified by Ni-NTA affinity chromatography after being expressed in E.coli,and the acivity of D-Alanyl-D-Alanine ligase was identified by D-amino oxidase.Herein,the method to detect D-Alanyl-D-Alanine ligase activity was established to be used for screening of its inhibitory compounds.In the other hand,purified Ddl A protein was used to prepare the polyclonal anti-Tb-Ddl A antibody which was applicaple for the detection of Sm-Ddl A in Mycobacteria Smegmatis that is regulated by down-regulation of antisense RNA.2.The effect of M.smeg-2395 on mycobacterail growth by down-regulation of antisense RNA.M.smeg-2395,a homologous sequence of Rv2981 c in Mycobacterium Smegmatis,was used to construct the antisense RNA to down regulate the ddl A expression in Mycobacterium Smegmatis strain.The growth curve of Mycobacterial strains,which was interrupted by antisense RNA of Sm-ddl A induced by doxycycline,was plotted according to cell absorbance at 595 nm versus growth time;The changes of bacterial morphology was observed for the down-regulated strains;The Ddl A protein expression was detected by polyclonal anti-Tb-Ddl A antibody in Mycobacterium Smegmatis.The obtained results as follows:1.The expression of Tb-Ddl A.The vector of p Cold-Tb-ddl A had been constructed to express Tb-ddl A in vitro.In this study,we successed in obtaining the purified Tb-Ddl A protein,and establishing an ELISA method to identify the activity of D-alanyl-D-alanine ligase by D-amino oxidase.The opitimal reaction factors had been determined as follows: substrate concentration of alanine was 10mmol/ml,100 mmol HEPES(p H 8.0)with 5 mmol/ml ATP was used as reaction buffer.The anti-Tb-Ddl A polyclonal antibody had been gained,and the titer and specificity of the antibody had been confirmed by enzyme linked immuno adsorption test and immunoblotting.2.The observation of the growth inhibition.p Mind-Sm-ddl A-As had been constructed and was introduced in Mycobacterium Smegmatis of wild type.So the down-regulated Mycobacteria strains by Sm-ddl A antisense RNA were successfully obtained in this study.The down-regulated expression of p Mind-Sm-ddl A-As is induced by doxycycline.Herein,we plotted a growth graph by the cell absorbance at 595 nm versus growth time.The results indicated that the cell growth was inhibited by Sm-ddl A antisense RNA,and the optimal induction conditions were doxycycline concentration of 20ng/ml,and incubation time of around 36 hours.3.The observation of cell morphology and the detection of deficient Ddl A in Mycobacterium Smegmatis.The total protein of p Mind-Sm-ddl A-As antisense down-regulation expression strains was extracted,and detected by anti Tb-Ddl A polyclonal antibody which was made in the lab.The results showed that the expression of Ddl A protein got down-regulated when the 20ng/ml doxycycline was introduced in and cultivation time was around 36 h.The results of transmission electron microscope indicated that the most of cell's morphology,which was regulated by p Mind-Sm-ddl A-As antisense RNA,got extended,the density of content inside the cell became heterogeneous.The possible research in the future:1.To screen the inhibitory compounds to target on Tb-Ddl A.2.To extract peptidoglycan of the strains regulated by p Mind-Sm-ddl A-As antisense RNA by HPLC.The extracted peptidoglycan will be cultured with RAW264.7 macrophages to observe whether the deficient peptidoglycan affect the infection occurred between host and Mycobaceria.3.To detect the antibiotic sensitive to anti-TB drugs for the strains which are regulated by down-regulation of antisense RNA.4.To generate and screen a large number of s RNAs,which are regulary RNA in prokaryotes,as possible and potential anti-Tuberculosis drugs.