Generation Of A Monoclonal Antibody Against Mycobacterium Tuberculosis Rv3295 Protein And Characterization Of Its Epitopes

The pathogenic microorganism of Mycobacterium tuberculosis is a harmful and serious threat for the human being. It has attracted scientists’ attention for a long time and a lot of researches have been done. There are lots of transcriptional regulators in Mycobacterium tuberculosis.Large network and sub-network of transcriptional regulators control different gene expression level in different physiological conditions aiming that the bacteria can adapt to bad environment and maintain normal metabolism which may be the target of drug.Until now,we have little know about the transcription regulators of Mycobacterium tuberculosis.Rv3295 protein belongs to TetR family transcription regulation, TetR family involves in the metabolism of bacteria, the carriage of small molecules, the generation of antibiotics and other physiological processes which plays an important regulatory role.rv3295 gene(683bp) was PCR-amplified using the genomic DNA of Mycobacterium tuberculosis H37 Rv as template and cloned to pET-22 b expression vector to construct recombinant plasmid pET22b-rv3295. The recombinant plasmid was sequenced and then was transformed into E. coli BL21(DE3). Rv3295 protein was correctly expressed at a final concentration of 1 mM IPTG under 30 ℃. Recombinant protein with 25 ku molecular weight was analyzed and confirmed by SDS-PAGE. High purity of Rv3295 protein was purified by affinity chromatography. Western-Blot confirmed that the recombinant protein Rv3295 with His-tag was successfully expressed.The recombinant protein Rv3295 was used as antigen to immune BALB / C mice for three times. One monoclonal antibody named Rv3295-1F7 was screened by cell fusion. Western-blot and subtype assay were applied to identify the specificity and subtype of Rv3295-1F7. The results showed that the subtype of Rv3295-1F7 was IgG1 with the κ light chain and this monoclonal antibody can be used for Rv3295 protein function study in the future.25 positive phage clones were screed using phage random peptide library panning Rv3295-1F7 monoclonal antibody for 3 rounds. Sequencing results showed that 6 phage clones had consensus sequences LTIHMDNHGPHR, and 2 had SWFGATGVGPHR, which shared similarity with amino acid 18-29 and 107-119 of Rv3295 protein in Mycobacterium tuberculosis. Phage-based ELISA and antigen competitive inhibition test confirmed that the phage displayed epitopes were similar with the natural epitopes of Rv3295 protein in Mycobacterium tuberculosis, therefore, they can be used to detect Mtb and develop epitope vaccines.