Cloning, Expression And Characterization Of Rv3717 Gene From M.Tuberculosis

Mycobacterium tuberculosis (M.tuberculosis) is one of major global pathogens and its threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, and it serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents,so it is difficult to develop new antimicrobial agents.Ethambutol (EMB) is one of the first-line antitubercular drugs. It was found that when the drug EMB was given to M. tuberculosis, the gene Rv3717 predicted possessing the fuction of hydrolyzing peptidoglycans up-regulated its expresstion, which could hydrolyze the peptidoglycan leading to the autolysis of the cell.Peptidoglycan is the important component of the core structure in the cell wall of M. tuberculosis. At present the understanding of the cell wall autolysin in mycobacteria (peptidoglycan hydrolase) is limited. The gene cwlB of bacillus subtilis is one of the known cell wall autolysins. The sequence of the gene was compared with the gene sequence of M. tuberculosis by BLAST, and we found two homologous gene Rv3915 and Rv3717 that were predicted their protein expresstion product possibly had the enzyme activity of peptidoglycan hydrolase. The research showed the gene Rv3915 was successfully cloned and expressed and had the activity of peptidoglycan hydrolase. Whether or not the gene Rv3717 had the same enzyme activity was the concern in this study.If the gene Rv3717 was proved having the enzyme activity of peptidoglycan hydrolase, we could provide certain theoretical evidence for the specific antitubercular mechanism of EMB and the development and research of new drugs.Objective: The amplification of the target gene Rv3717; Constructing the cloning plasmid pMD18-Rv3717; Constructing the expression plasmid pET29b-Rv3717 and pET16b-Rv3717; Optimizing the expression of the target protein; The purification and fuction identification of the target protein.Methods: The amplification of the target gene using the genome DNA of M. tuberculosis for the template and using the PCR technology; Constructing the cloning plasmid pMD18-Rv3717 using the cloning vector pMD18-T and the target gene with the connection type"A-T"; After the amplified target fragment was correctly sequenced, the carrier and the target fragment were connected by double-enzyme cleavage in order to construct the expression plasmid; Optimizing the expression of the target protein using different vectors and different host bacteriums; The expression of the target gene was detected using SDS-PAGE and Western blotting method; The purification of the target protein using Ni²⁺ affinity chromatography column and the dectection of the protein fuction using denatured polyacrylamide gel containing M. smegmatis cell wall.Results: The cloning plasmid pMD18-Rv3717 was constructed; The non-mutated bases expression plasmid pET29b-Rv3717 and pET16b- Rv3717 were constructed after the amplified target fragment was correctly sequenced. The expression plasmid pET29b-Rv3717 was expressed very lowly in the used various kinds of host bacteria. The expression plasmid pET16b-Rv3717 was highly expressed in E.coli BL21(DE3), but the vast majority of the target protein existed in the form of inclusion body and the soluble protein in liquid supernatant was less. The target protein in liquid supernatant was purified by Ni²⁺ affinity chromatography column and we got the purification content of the target protein containing a little miscellaneous protein. The enzyme activity of the target protein purified was not found.Conclusions: The gene Rv3717 of M. tuberculosis was cloned using molecular cloning technique and was overexpressed using the expression plasmid pET16b in E.coli BL21(DE3)and the vast majority of the target protein existed in the form of inclusion body. The thesis discussed the relation between the target gene Rv3717 and peptidoglycan hydrolase and the peptidoglycan hydrolase activity of target protein was not found, but it provided the methodology reference and material base for further exploring the relationship.