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Expression and purification of recombinant antigens of Mycobacterium tuberculosis for application in serodiagnosis.

Abstract：

:Accurate diagnosis is essential for the treatment, prevention, and control of tuberculosis. Poor specificity of the tuberculin skin test in BCG-vaccinated populations and constraints to implementation of PCR and CMI-based diagnostic assays in developing countries warrant development of easy-to perform robust serological tests. Due to great heterogeneity in humoral response in TB patients, it will be necessary to include several antigens in any diagnostic assay to achieve useful levels of sensitivity and specificity. This needs production of recombinants, soluble versions of mycobacterial antigens in high yields. We have cloned, expressed, and purified a number of mycobacterial proteins in Escherichia coli. This paper describes the expression and purification of four promising sero-reactive proteins namely, ESAT6, CFP10, MTC28, and 14-kDa antigen of Mycobacterium tuberculosis. The protocol involves regulated and slow expression of proteins by using a T7 promoter-based expression vector for obtaining soluble protein followed by a three-step column chromatography procedure employing media with high binding capacity and flow characteristics. The yields of these proteins obtained were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of TB patients (smear positive, smear negative, and extra-pulmonary categories) and in combination with other immunodominant antigens will be useful in increasing the sensitivity to detect M. tuberculosis specific antibodies.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Kulshrestha A,Gupta A,Verma N,Sharma SK,Tyagi AK,Chaudhary VK

doi

10.1016/j.pep.2005.04.014

keywords:

subject

Has Abstract

pub_date

2005-11-01 00:00:00

pages

75-85

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(05)00151-8

journal_volume

44

pub_type

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