Cloning, expression, identification and bioinformatics analysis of Rv3265c gene from Mycobacterium tuberculosis in Escherichia coli

OBJECTIVE@#To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E. coli) under optimistic conditions, obtain and identify protein expressed, analyze the structure and characteristics of the protein using bioinformatics methods for future applications.@*METHODS@#Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction, and was cloned into the pET-30a vector after purification and recovery. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3), and then purified and identified by western blotting. The essential physical-chemical properties of the protein were predicated by bioinformatics tools, including subcellular location, secondary structure, domains, antigenic epitopes, etc. Tertiary structure of the protein based on homology modeling was established, while multi-sequence homological alignment and phylogenetic analysis were proformed.@*RESULTS@#The recombinant protein was obtained in soluble fraction from expression system in E. coli BL21(DE3) carrying pET30- Rv3265c plasmid, and Rv3265c gene was expressed correctly. Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices, located outside of membrane. Secondary structure analysis revealed it contained α-helix, extended strand and random coil, 46.8%, 14.6%, 38.6%, respectively. Furthermore, it possessed six potential antigenic epitopes, one glycosyl transferase domain. A simple three-dimensional model of this protein was constructed by Swiss-model sever. Both sequences and structures were conservative and especial either in gene or in protein.@*CONCLUSIONS@#Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine. The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.

Prokaryotic expression, identification and bioinformatics analysis of fbpB-esxA fusing gene from Mycobacterium tuberculosis

OBJECTIVE@#To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis (MTB), express the encoded fusing protein in Escherichia coli (E. coli), identify protein acquired, and predict the structure and function of the protein utilizing methods of bioinformatics.@*METHODS@#fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR. The fbpB-esxA fusing gene ligated by (Gly(4)Ser)(3) linker was gained by means of Gene Splicing by Overlapping Extension PCR (SOE-PCR), and fusing gene was cloned into expression vector pET-30a. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3). The protein was identified by Western blot using anti-HIS antibody. Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics.@*RESULTS@#The DNA sequences of fbpB-esxA were identical with that published by GenBank. The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E. coli BL21(DE3) under the induction of IPTG. Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes.@*CONCLUSIONS@#The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag. Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly. The existence of linker doesn't affect immunogenicity of Ag85B and ESAT-6. It will allow for characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.