Construction And Expression Of Antisense RmlC Expression Vector

Objective: Mycobacterium Tuberculosis is one of the most serious diseases to people health. Recently, the number of the people who caused the disease rises again. The main reason is the emergence of multi-drug resistant strains and the unefficient anti-TB drugs, which lead the death rate increased rapidly. Thus, there is the urgent need to develop new drugs for the disease. Mycobacterium tuberculosis is the pathogen of tuberculosis. The cell wall is essential for the existing and reproduction of the Mycobacterium tuberculosis and it is the ideal target for development of new drugs.The mycobacterial cell wall core consists of three interconnected'macromolecules': the outermost, mycolic acids are esterified to the middle component arabinogalacan (AG).AG is attached to the peptidoglycan via a linker disaccharide,α-L-rhamnosyl-(1-3)-α-D-N-acetyl- glucosaminosyl- 1-pos-phate. The structure of the mycobacteria cell wall strongly suggests that the linker disaccharide is an important component. The L-rhamnosyl residue of linker disaccharide is provided with a sugar donor, dTDP- rhamnose. The biosynthetic pathway of dTDP-rhamnose consists of four-step reactions fromα-D-glucose-l-phosphate and TTP to dTDP- rham- nose through three intermediates, and four reactions are catalyzed by four enzymes,α-D-glucose-1-phosphate thymidylyltransferase (RmlA), dTDP- D-glucose-4, 6-dehydratase (Rm1B), dTDP-4-keto-6-deoxy-glucose-3, 5- epimerase (RmlC), and dTDP-6-deoxy-L-lyxo-4-hexulose reductase (dTDP -4-keto-L-rhamnose reductase) (Rm1D), respectively. The findings and thefacts that there is no salvage pathway for the formation of dTDP-rhamnose and L-rhainnosyl residues are not found in humans support the enzymes involved in dTDP-rhamnose biosynthesis are important targets for the development of new anti-tuberculosis drugs. Thus it is important to directly demonstrate that enzymes involved in dTDP-rhamnose formation are indeed essential. It has been proved that the Rm1C (dTDP-4- keto-6- deoxy-D-glucose 3, 5-epimerase) are essential for growth of mycobacteria by using gene knock out strategy.To demonstrate the effect of low RmlC on the cell wall, our objectives in this study are: (1) to clone rmlC gene to antisense expression vector pMind-rmlC-AS and electroporate pMind-rmlC-AS to mc~2155 competent cells. (2) To clone rmlC gene to expression vector pET29b and express RmlC protein in Ecoli BL21(DE3). (3) To purify RmlC protein and immu- nize the BALB/C mouse to prepare anti-RmlC antibody. (4) To test the effect of rmlC antisense RNA on growth of mc~2 155.Results:1. Constructed of antisense expression vector pMind-rmlC-AS The rmlC nucleotide sequence was acquired from TIGR (http://www. tigr.org/tdb/) data, One set of primers was designed and site BamHI and NdeI site were added to 5'end of upstream primer and downstream primer respectively. The purified PCR product was ligated into pSTBlue I plasmid, and E.coli NovaBlue competent cells were transformed with the ligation reaction products. Recombinant plasmid pSTBlue 1-rmlC was confirmed by digestion of BamHI and NdeI and sequencing. pSTBlue 1-rmlC was digested by BamHI and NdeI, rmlC gene was ligated into the BamHI and NdeI sites of pMind, resulting in rmlC antisense expression vector pMind-rmlC-AS. pMind-rmlC-AS was electroporated to mc~2155 competent cells.2. Constructed of expression vector pET29b-rmlCThe primers for rmlC gene amplification were designed and site NdeI and XhoI site were added to 5'end of upstream primer and downstream primer respectively. The purified PCR product was ligated into pMD18-T vector. Recombinant plasmid pMD18-rmlC was confirmed by enzyme digestion and sequencing. pMD18-rmlC was digested by NdeI and XhoI,and the rmlC gene was ligated into the NdeI and XhoI sites of pET29b resulting in pET29b-rmlC. The C-terminus of RmlC protein was fused with His-tag (6 Histidines).3. Expression, purification and identification of the RmlC protein BL21(DE3) competent cells were transformed with pET29b-rmlC.RmlC protein was expressed at 1 mM IPTG at 37°C for 4 hours. Induced BL21(DE3)/pET29b-rmlC cells were sonicated and both supernate and pellet fractions were analyzed by SDS-PAGE. RmlC protein was purified by Ni-NTA column. Identification of the RmlC protein by Western blot showed that RmlC protein was expressed in BL21(DE3).4. Preparation and identification of anti-RmlC polyclonal antibodyBALB/C mice were immunized with purified RmlC protein every two weeks for four times. The anti-RmlC polyclonal antibody was collected and identified by ELISA. The result showed its efficiency at 1:6400.5. Expression of pMind-rmlC-AS in mc~2155pMind-rmlC-AS was electroporated to mc~2155 competent cells. The mc~2155/pMind-rmlC-AS and mc~2155 were induced by tetracycline (Tc) at the concentration of 10 ng/ml and 20 ng/ml. The absorbance measured at 600 nm showed the pMind-rmlC-AS/mc~2155 grew slowly than mc~2155 obviously. The RmlC protein expressed mc~2155/pMind-rmlC-AS and mc~2155 induced by Tc at the concentration of 10 ng/ml and 20 ng/ml was detected by the prepared anti-RmlC polyclonal antibody. The results showed no distinct difference between mc~2155/pMind-rmlC-AS and mc~2155. Therefore, it is necessary to optimize Tc concentration for induction.Conclusions:In this study, (1) we cloned rmlC gene from the Mycobacterium smegmatis mc~2155 genomic DNA. (2) We constructed the pMind-rmlC-AS antisense expression plasmid. (3) We constructed the pET29b-rmlC expression plasmid. (4) We purified the expressed protein and prepared the anti-RmlC polyclonal antibody. (5) We electroporated pMind- rmlC to mc~2155 competent cells and induced mc~2155/pMind-rmlC-AS and mc~2155 induced by Tc at the concentration of 10 ng/ml and 20 ng/ml and the RmlC protein was detected by Western blot.This rmlC antisense RNA expression system will be useful to studythe effect of lacking RmlC on mycobacterial cell wall.