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Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens.

Mahmoudi S, Mamishi S, Ghazi M, Hosseinpour Sadeghi R, Pourakbari B - Iran J Microbiol (2013)

Bottom Line: ESAT-6 andCFP-10 proteins were purified by Ni-NTA affinity chromatography and were detected by anti- ESAT-6 and anti -CFP-10 antibodies.ESAT-6 andCFP-10 genes were successfully expressed and purified.Anti- ESAT-6 and anti-CFP-10 antibodies were produced after induction of immunization against purified ESAT-6 andCFP-10 proteins in rabbit.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Infectious Disease Research Center, Tehran University of Medical Sciences, Tehran, Iran.

ABSTRACT

Background and objectives: ESAT-6 (6-kDaearly secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) have been described as dominant antigens recognized by T-cells and considered as virulence factors in Mycobacterium tuberculosis. The aim of this study was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M. tuberculosis in soluble form.

Materials and methods: ESAT-6 andCFP-10 genes were amplified by PCR, cloned into pET32a (+) vector, and overexpress-ed using isopropyl-beta-D-thiogalactopyranoside in E. coli BL21 (DE3). ESAT-6 andCFP-10 proteins were purified by Ni-NTA affinity chromatography and were detected by anti- ESAT-6 and anti -CFP-10 antibodies.

Results: ESAT-6 andCFP-10 genes were successfully expressed and purified. Anti- ESAT-6 and anti-CFP-10 antibodies were produced after induction of immunization against purified ESAT-6 andCFP-10 proteins in rabbit.

Conclusion: In this study, we cloned, expressed and purified sufficient amounts of ESAT-6 andCFP-10 and it would be tested for the development of diagnostic kit for M. tuberculosis in future.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of recombinant CFP-10 and ESAT-6 proteins; lane 1, purified ESAT-6 proteins; lane 4, 5, purified CFP-10 protein; lanes 2,3,6,7, negative control; lanes 8, prestained protein molecular weight marker #26612 Thermo Scientific.
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Figure 3: Western blot analysis of recombinant CFP-10 and ESAT-6 proteins; lane 1, purified ESAT-6 proteins; lane 4, 5, purified CFP-10 protein; lanes 2,3,6,7, negative control; lanes 8, prestained protein molecular weight marker #26612 Thermo Scientific.

Mentions: ESAT-6 andCFP-10 proteins were purified and detected by anti- ESAT-6 and anti -CFP10 antibodies. Western blot analysis confirmed the presence of recombinant ESAT-6 andCFP-10 protein (Fig. 3).


Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens.

Mahmoudi S, Mamishi S, Ghazi M, Hosseinpour Sadeghi R, Pourakbari B - Iran J Microbiol (2013)

Western blot analysis of recombinant CFP-10 and ESAT-6 proteins; lane 1, purified ESAT-6 proteins; lane 4, 5, purified CFP-10 protein; lanes 2,3,6,7, negative control; lanes 8, prestained protein molecular weight marker #26612 Thermo Scientific.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385163&req=5

Figure 3: Western blot analysis of recombinant CFP-10 and ESAT-6 proteins; lane 1, purified ESAT-6 proteins; lane 4, 5, purified CFP-10 protein; lanes 2,3,6,7, negative control; lanes 8, prestained protein molecular weight marker #26612 Thermo Scientific.
Mentions: ESAT-6 andCFP-10 proteins were purified and detected by anti- ESAT-6 and anti -CFP10 antibodies. Western blot analysis confirmed the presence of recombinant ESAT-6 andCFP-10 protein (Fig. 3).

Bottom Line: ESAT-6 andCFP-10 proteins were purified by Ni-NTA affinity chromatography and were detected by anti- ESAT-6 and anti -CFP-10 antibodies.ESAT-6 andCFP-10 genes were successfully expressed and purified.Anti- ESAT-6 and anti-CFP-10 antibodies were produced after induction of immunization against purified ESAT-6 andCFP-10 proteins in rabbit.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Infectious Disease Research Center, Tehran University of Medical Sciences, Tehran, Iran.

ABSTRACT

Background and objectives: ESAT-6 (6-kDaearly secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) have been described as dominant antigens recognized by T-cells and considered as virulence factors in Mycobacterium tuberculosis. The aim of this study was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M. tuberculosis in soluble form.

Materials and methods: ESAT-6 andCFP-10 genes were amplified by PCR, cloned into pET32a (+) vector, and overexpress-ed using isopropyl-beta-D-thiogalactopyranoside in E. coli BL21 (DE3). ESAT-6 andCFP-10 proteins were purified by Ni-NTA affinity chromatography and were detected by anti- ESAT-6 and anti -CFP-10 antibodies.

Results: ESAT-6 andCFP-10 genes were successfully expressed and purified. Anti- ESAT-6 and anti-CFP-10 antibodies were produced after induction of immunization against purified ESAT-6 andCFP-10 proteins in rabbit.

Conclusion: In this study, we cloned, expressed and purified sufficient amounts of ESAT-6 andCFP-10 and it would be tested for the development of diagnostic kit for M. tuberculosis in future.

No MeSH data available.


Related in: MedlinePlus