Preparation Of Anti-GlmU Antibody And Detection Of GlmU Antisense RNA Expression

The cell wall of mycobacterial has unique structure. The core of the cell wall consists of peptidoglycan, arabinogalactan, and mycolid acid. The mycolic acid and arabinogalactan are attached to peptidoglycan via the disaccharide linker (L-rhamnosyl-N-acetyl-glucosaminyl-phosphate). UDP-N-acetylglucosamine (UDP- GlcNAc), the nucleotide-activated form of N-acetylglucosamine, is a donor of GlcNAc in disaccharide linker. GlmU protein encoded by glmU gene is bifunctional enzyme: glucosamine-1-phosphate acetyl transferase and N-acetylglucosamine-1-phosphate uridyltransferase, which catalyze two sequential steps in UDP-GlcNAc biosynthesis. The research data from Zhang Wenli in our laboratory demonstrate that glmU gene is essential for mycobacterial growth. Therefore, GlmU is a potenial target to develop anti-tuberculosis drugs.To investigate the effect of GlmU enzyme on the structure and composition of polysaccharides in mycobacterial cell wall and establishe the cell model to screen GlmU inhibitors, a tetracycline inducible expression vector of glmU antisense RNA, pMind-glmU-AS has been constructed in our laboratory. To optimize the concentra -tion of tetracycline to control expression of glmU antisense RNA, it is necessary to detect the expression of GlmU enzyme in M.smegmatis mc2155 cells carrying pMind-glmU-AS by anti-GlmU protein antibody.The Objectives of this study are: (1) To amplify glmU gene from the genomic DNA of M. smegmatis mc2155 by polymerase chain reaction (PCR), construct a cloning vector pMD18-glmU and sequcence glmU gene in pMD18-glmU. (2) To subclone glmU gene to an expression vector pET29b to construct pET29b-glmU and overexpress GlmU protein in BL21(DE3) carrying pET29b-glmU. To purify GlmU protein by affinity chromatography and identify GlmU protein by SDS-PAGE and Western blotting.(3) To prepare anti-GlmU polyclonal antibody by injecting BalB/C mice with purified GlmU protein. (4) To induce expression of glmU antisense RNA by different concentration of tetracyclin and detect GlmU protein in M. smegmatis mc2155 cells carrying pMind-glmU-AS by Western blotting.Followings are results I acquired in this study:1. Amplification of glmU gene and construction of pMD18-glmUThe sequence (1449 bp) of M. smegmatis glmU gene was acquired from M. smegmatis mc~2155 genome by blasting M. tuberculosis H37Rv GlmU protein against mc~2155 genome database of TIGR. One set of primers were designed based on the sequence of glmU gene, and Nde I site and Xho I site were added to 5'end of the upstream and downstream primer, respectively. The glmU was amplified from mc2155 genomic DNA by using a high-fidelity DNA polymerase, LA Taq DNA polymerase. The purified PCR product was ligated into pMD18-T vector, resulting in pMD18-glmU plasmid and glmU gene was sequenced confirming glmU gene with correct bases.2. Construction of expression vector pET29b-glmUpMD18-glmU was digested by Nde I and Xho I, then glmU gene fragment was purified and ligated into the Nde I and Xho I sites of vector pET29b to generate pET29b-glmU. The C-terminus of GlmU protein was fused with histidine tag in pET29b vector.3. Expression of GlmU protein in E.coli BL21(DE3) and purification of GlmU proteinpET29b-glmU were transformed into BL21(DE3) competent cells. BL21(DE3) cells carring pET29b-glmU were induced with IPTG and total proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE. The results showed that soluble GlmU protein was produced in BL21(DE3) cells.GlmU protein was purified by histidine-Ni2+affinity chromatography. The elution fraction 1 (1 ml) was quantified as 0.635 mg/ml by coomassie brilliant blue method. The purified GlmU protein was identified by SDS-PAGE and Western blotting.4. Preparation of anti-GlmU polyclonal antibodyPurified GlmU was prepared with particular means for injecting BalB/C mice. Antiserum was separated and assayed for antibody titer as 1:51200 by enzyme-linked immunosorbent assay. Only GlmU protein of mc2155 total protein was detected by antiserum followed by antimouse-IgG-conjugated with alkaline phosphatase. The result showed anti-GlmU polyclonal antibody had high specificity.5. Expression of glmU antisense RNA induced by tetracycline and detection of GlmU protein in mc2155 carrying pMind-glmU-AS.The mc2155 carrying pMind-glmU-AS was induced by different concentration of tetracycline, and GlmU protein was detected in the mc2155 carrying pMind-glmU-AS by Western blotting. The results showed that tetracycline of 20ng/ml has obviously inhibited the growth rate of mc2155 carrying pMind-glmU-AS, but the amount of GlmU protein did not change compared to that of uninduced mc2155 carrying pMind-glmU-AS.Conclusions:In this study, we constructed pET29b-glmU expression vector and overexpressed considerable soluble recombinant GlmU protein in E.coli BL21(DE3). We prepared anti-GlmU polyclonal antibody with the purified GlmU and utilized anti-GlmU polyclonal antibody to detect GlmU protein in the mc2155 carrying pMind-glmU-AS. We tried to explore the correlation between the expression level of GlmU protein and growth rate of mc~2155 carrying pMind-glmU-AS. The expression of glmU antisense RNA will be utilized to study the correlation between GlmU enzyme activity and strtucture of mycobaterial cell wall.