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Cells
HeLa cells (1×106 cell/ml) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Lonza) supplemented with 10% fetal calf serum (FCS; Lonza), 2 mM glutamine and 1% antibiotics (penicillin and streptomycin; Lonza), and incubated overnight at 37 C. Nonadherent cells were removed by washing with PBS (Lonza). The cells were then incubated for a further 24 hours in fresh DMEM. The cells were allowed to grow to a confluency of 75-80%.
Monocyte-derived macrophages (MDMs), for use in biological activity assays, were obtained as follows: Firstly, human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque™ PLUS (GE Healthcare) density gradient centrifugation from heparinised blood of healthy donors (South African National Blood Bank), as described previously (Khati et al., 2003). The PBMCs were suspended in RPMI medium containing 20% FCS, seeded into 150-cm2 tissue culture flasks (Corning, Adcock Ingram) and incubated for 2 hours at 37 C. Subsequently, non-adherent cells were removed, and the remaining adherent cells (monocytes) were washed in PBS (Lonza) and incubated for a further 24 hours in fresh DMEM. The monocytes were then harvested, suspended in XVIVO 10 (BioWhittaker) supplemented with 2% autologous serum and re-seeded into tissue culture flasks. Following seven days of differentiation, the resulting MDMs were typed by flow cytometry using an antibody directed against the CD68 cell surface receptor (R&D Systems) and detected with a secondary FITC-conjugated antibody (Kirkegaard and Perry Laboratories (KPL). Only cultures in which MDMs represented at least 95% of the cells were used.
Expression of CFP-10
E. coli BL21 (DE3) cells (Novagen) were transformed with the T7 promoter-based expression plasmid pMRLB46 (Megan Lucas, Colorado State University), expressing fulllength CFP-10 fused to an N-terminal hexahistine tag. Transformed cells were plated onto LB agar plates containing 100 μg/ml ampicillin (Fermentas) and left to grow overnight. Thereafter, a single colony was picked and grown in 5 ml of LB medium and 100 μg/ml ampicillin at 37°C in a shaking incubator at 200 rpm overnight. The overnight cultures were inoculated into 2-L Erlenmeyer flasks, containing 1 L of LB medium and 100 μg/ml ampicillin, at 37°C in a shaking incubator at 200 rpm. Expression of the recombinant protein was induced in mid-exponential phase cultures, corresponding to an optical density (OD) at 600 nm of 0.6, by the addition of isopropyl-1-thio-β-Dgalactopyranoside (IPTG; Sigma) to a final concentration of 0.4 mM. The cultures were harvested 4 hours after induction by centrifugation at 6 500 rpm for 20 minutes at 4°C.
The cell pellets were stored overnight at -80°C.
Purification and solubilisation of CFP-10
Cell pellets from induced cultures were suspended in 10 ml of Buffer A and lysed by sonication on ice at 60% power output for 3 minutes. This step was repeated three times, after which the lysate was centrifuged at 14 000 rpm for 15 minutes. The supernatant was recovered and loaded onto a Ni-NTA column (BioRad), which had been preequilibrated with Buffer A, by rotation overnight at 4°C. The Ni-NTA column was washed once with 100 ml of Buffer A. The urea was removed by six washes with 10 ml of Buffer A, in which the urea concentration was decreased from 6 M to 1 M, followed by two washes with 20 ml of Buffer B. Finally, CFP-10 was eluted in a single step with 10 ml of Buffer B containing 300 mM imidazole. The purity of the eluted protein was assessed by electrophoresis on a 17% SDS-PAGE gel and stained with Coomassie brilliant blue R- 250 (0.25% Coomassie Blue R-250, 50% Methanol and 10% acetic acid).
Chapter 1. LITERATURE REVIEW
1.1 – MYCOBACTERIUM TUBERCULOSIS (MTB)
1.2 – DIAGNOSTIC TOOLS AVAILABLE FOR MTB
1.3 – APTAMERS AS A DIAGNOSTIC TOOL FOR MTB INFECTION
1.4 – CFP-10/ESAT-6 HETERODIMER
1.5 – SCOPE AND OBJECTIVES
1.6 – STRUCTURE OF THESIS
Chapter 2. EXPRESSION AND PURIFICATION OF CFP-10 AND ESAT-6
SUMMARY
2.1 – INTRODUCTION
2.2 – MATERIALS AND METHODS
2.3 – RESULTS
2.4 – DISCUSSION
Chapter 3. ISOLATION AND CHARACTERISATION OF APTAMERS TARGETING THE CFP-10/ESAT-6 HETERODIMER
SUMMARY
3.1 – INTRODUCTION
3.2 – MATERIALS AND METHODS
3.3 – RESULTS
3.4 – DISCUSSION
Chapter 4. EVALUATION OF LIMIT OF DETECTION AND DETECTION OF MTB ANTIGENS IN CLINICAL SPUTUM SAMPLES USING APTAMERS
SUMMARY
4.1 – INTRODUCTION
4.2 – MATERIALS AND METHODS
4.3 – RESULTS
4.4 – DISCUSSION
Chapter 5. GENERAL DISCUSSION AND CONCLUSION
5.1 – SUMMARY OF FINDINGS
5.2 – IMPLICATIONS OF THIS STUDY IN RELATION TO CURRENT TB DIAGNOSTICS
5.3 – CHALLENGES AHEAD
5.4 – FUTURE WORK
REFERENCES
