Cloning, Expression And Partial Characterization Of MSMEG₁401 Gene From Mycobacterium Smegmatis

Tuberculosis (TB) is a kind of ancient disease, endangering the human being's health seriously, is also the public hygiene problem and social problems of the global concern. Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. M.tuberculosis had once been controlled, but now it poses a major public health problem. The emergency of multi-drug resistant strains and co-infection of M. tuberculosis with HIV are the two greatest factors that have contributed to the global resurgence of TB. Thus, more efficient anti-TB drug are desperately needed.The mycobacterial cell wall is a unique structure for mycobacterial survival and growth in the host. The core of mycobacterial cell wall consists of inner layer peptidoglycan (PG), middle layer arabinogalactan (AG) and outer layer mycolic acid. AG is an important structure to connect PG and mycolic acid and to keep the integrity of the cell wall. Therefore, AG is an ideal target to develop new effective anti-TB drugs. And through the clarification to existing drug's mechanism of action, we can find the critical proteins in the relevant metabolic process and ascertain them as the potential drug targets.Ethambutol (EMB) is the most common first-line anti-TB drug, it inhibits the growth of M.tuberculosis in reproduction period, and hasn't manifest obvious drug resistance. An experimental result of Mike McNeil professor from Colorado State Universities'microorganism specialty indicated that after adding EMB in the culture medium of M.tuberculosis, the arabinan of cell wall lost in short period, making that the ratio of arabinose to galactose vary obviously. To find the acting mechanism of EMB and its relationship with the metabolic variation of cell wall's polysaccharides, Zhan Yaoyao has used the methods of two-dimensional electrophoresis and mass spectrum found out the different expressed proteins between wild M.smegmatis mc2 155 and EMB-treated M. smegmatis mc² 155 previously. (Because the cell wall's constitution of M.smegmatis mc² 155 is similar to M.tuberculosis, moreover it grows rapidly and has no pathogenicity, so we use it as the modeling bacterium of M.tuberculosis ).The objectives of this study: based on the two-dimensional electrophoresis and mass spectrum's results of Zhan Yaoyao, we search relevant proteins'gene sequence from NCBI gene sequence database, design primers and adopt RT-PCR ( reverse transcriptase polymerase chain reaction ) to detect the expressive variation of genes which are corresponding to different expressed proteins from wide and EMB-treated M.smegmatis mc2 155, if expressive level enhance obviously, we should clone and express the corresponding gene in M.smegmatis mc2 155 to raise the relevant gene's expression artificially. Ultimately we will use HPLC ( high performance liquid chromatography ) to detect whether the ratio of cell wall's arabinose content to galactose content varies, if the ratio reduces, it indicates that the corresponding gene's function is close to the metabolism of cell wall's polysaccharides, the result can lead us in the further study and research.Followings are the experimental content and results we got in this study:1. Selection of the objective gene.According to the mass spectrum's results of Zhan Yaoyao's, we searched corresponding genes'sequences from NCBI protein and gene sequence databases, designed primers and made RT-PCR experiment, we selected 12 corresponding genes which varied notablely in mass spectrum's results to carry out RT-PCR ultimately. Among the total, there are four genes'expression is up-regulated and eight genes'expression is down-regulated, the four genes are accA,tpx,tuf,bfr; the eight genes are groEL,cys,mtrA,hisA,fdxA,gpd,gk,pst. According to the result analyzed by Labworks, we select the most notablely variable gene in up-regulated group as the objective gene to carry out cloning and expression.2. MSMEG₁401 gene was amplified from M.smegmatis mc2 155 genomic DNA by polymerase chain reaction (PCR).DNA sequence of MSMEG₁401 gene (1191bp) was acquired from NCBI gene database. One set of primers was designed based on the sequence, NdeI site and HindIII site were added to 5'end of upstream primer and downstream primer respectively. MSMEG₁401 gene was amplified from M.smegmatis mc2 155 genomic DNA by LA Taq DNA polymerase with high-fidelity.3. Cloned and sequenced MSMEG₁401 PCR product.MSMEG₁401 PCR product was ligated into the EcoRV site of pMD18-T plasmid to generate pMD18- MSMEG1401. MSMEG₁401 gene was sequenced and sequence was compared to the MSMEG₁401 gene from M.smegmatis mc2 155 database by Multalin ( http://prodes.toulouse. inra.fr/multalin/multalin.html ). Alignment result showed that cloned MSMEG₁401 gene is identical to the MSMEG₁401 gene in M.smegmatis mc2 155 database. There is no any mutation in the cloned MSMEG₁401 gene.4. Expression plasmid pVV2-MSMEG1401 was constructed.pMD18-MSMEG1401 was digested by NdeI and HindIII, MSMEG 1401 gene fragment was purified and ligated into the NdeI and HindIII sites of plasmid pVV2 resulting in pVV2-MSMEG1401. pVV2-MSMEG1401 was confirmed by digestion of restriction endonuclease BamHI and KpnI. The N-terminus of MSMEG₁401 protein was fused with His-tag ( 6 Hisitidines ) from pVV2-MSMEG1401. His-tag is used for detection of MSMEG₁401 protein by Western blotting.5. MSMEG₁401 protein was expressed in M.smegmatis mc2 155.pVV2-MSMEG1401 was electroporated into M.smegmatis mc2 155 competent cells and MSMEG₁401 protein was expressed at 37°C, the objective protein was detected by SDS-PAGE and Western blotting.6. Utilize HPLC to detect the ratio variation of arabinose content to galactose content in cell wall.Extracting the cell wall's polysaccharides of wild M.smegmatis mc2 155 and the M.smegmatis mc2 155 which has highly expressed MSMEG- 1401 protein,preparing monosaccharide of arabinose and galactose. In specified HPLC condition, we compare the ratio of arabinose content to galactose content in cell wall from the two strains have mentioned previous according to the area of peak.Conclusions:In this study, we cloned MSMEG₁401 gene by molecular cloning tech, and highly expressed MSMEG₁401 protein in M.smegmatis mc2 155. Ultimately we applied HPLC to detect the relationship between MSMEG 1401 gene and the metabolism of cell wall's polysaccharide, that will be the premise for the further investigation into the correlation of MSMEG₁401 gene and arabinose.