30470097, No. distributed among high G + C Gram-positive bacteria, and genome sequencing has uncovered examples in possesses five genes with significant homology to the of (Rv0867c), (Rv1009), (Rv1884c), (Rv2389c) and (Rv2450c) share a conserved segment, which encodes an Rpf-like domain of about 70 residues long[15]. More recently, the Rpf-like proteins of have been shown to stimulate the growth of extended-stationary-phase cultures of BCG[12]. Our previous study also showed that purified recombinant RpfD could stimulate the resuscitation of H37Ra[16]. These data suggest that the Rpf proteins can influence the growth of mycobacteria[17]. Surprisingly, all of the five individual deletion mutant strains Embelin showed growth kinetics similar to the wildtype strain, likely due to the redundancy[15],[18]. Bacteria with deletion of multiple genes (such as in resuscitation from the nonculturable state[18]. Sequence analysis suggests that at least some of these proteins are secreted and that all five proteins probably have extracytoplasmic functions[19], making them potential targets for recognition by the host immune system at the stage of reactivated disease. Therefore, these proteins have potential as novel diagnostic reagents and subunit vaccine candidates for control of TB. In this study, we described the expression and purification of recombinant RpfE proteins in (iRpfE) and (sRpfE) with regard to their NFBD1 immunogenic properties. MATERIALS AND METHODS Bacterial strains, plasmids and animals H37Rv and BCG were grown in Middlebrook 7H9 medium supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% oleic albumin dextrose catalase (OADC) enrichment (Becton Dickinson, NJ, USA) at 37C. The bacteria were grown to an optical density at 600 nm of 1 1 in roller bottles, divided into 1 mL aliquots in cryovials, and stored at -70C. DH5 and were grown on solid or Embelin in liquid Luria-Bertani medium. The expression vectors pPRO-EXHT (Invitrogen Life technologies, USA) and pDE22 (a shuttle secretory plasmid for into expression vectors Genomic DNA was isolated from H37Rv Embelin using a standard phenol/chloroform extraction protocol[20]. The gene was amplified from genomic DNA with a pair of primers which were designed based on the known DNA sequence (Tuberculist Accession No. Rv2450): 5-CCGGGATCCCATCACCATCACCATCACATGAAGAACGCCCGTACGACG-3, which contained an III site (underlined). The reactions were performed using rpolymerase (Takara, Dalian, China) in a final volume of 25 L. The thermal cycling program was performed in a thermo cycler (MJ Research, Watertown, MA, USA) and the conditions were as follows: 30 cycles of 30 sec at 94C, 30 sec at 58C, and 60 sec at 72C. The amplified product was digested with I and III, and then ligated to the corresponding sites of the expression vectors pPRO-EXHT and pDE22. Finally, both recombinant vectors were checked for the correct orientation and DNA sequence by sequencing in both directions (Invitrogen Life technologies, Beijing, China). The correct plasmids were designated as pPRO-EXHT-rpfE and pDE22-rpfE, respectively. Transformation Embelin of DH5 and DH5 and were prepared as previously described[16]. For electroporation, 1-2 L of pPRO-EXHT-rpfE and pDE22-rpfE plasmids were added to 0.4 mL of the competent DH5 and suspensions, respectively. The mixture was incubated on ice for 10 min and transferred into a 0.2 cm electrode gap electroporation cuvette (Bio-Rad, Hercules, CA, USA) and was subjected to a single-pulse electroporation of 25 F at 2.5 kV, with resistance set at 1,000 . After electrotransformation, the cuvettes were put back on ice for 10 min, and then the mixtures were transferred into 5 mL of LB broth. The culture was then incubated at 37C for 2 h followed by centrifugation at 3,000 for 10 min. DH5 cells were plated on LB agar plate containing 100 g/mL ampicillin, and cells were plated on LB agar plate containing 100 g/mL hygromycin. The plates were incubated at 37C until colonies became visible. Expression and purification of recombinant iRpfE in DH5 DH5 (pPRO-EXHT-rpfE) cells were grown in 200 mL of LB medium with shaking (100 for 10 min to.
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