Analysis of the shotgun expression library of the Mycobacterium tuberculosis genome for immunodominant polypeptides: potential use in serodiagnosis.
A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the lambdagt11 vector. DNA from a virulent M. tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides. beta-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis. The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%.
Bisen
Prakash S
PS
Department of Biotechnology, Madhav Institute of Technology and Science, Gwalior 474005, Madhya Pradesh, India. prakash_bisen@hotmail.com
Garg
Sanjay K
SK
Tiwari
Ram P
RP
Tagore
P Ravindra Nath
PR
Chandra
Ramesh
R
Karnik
Rucha
R
Thaker
Nimesh
N
Desai
Nirav
N
Ghosh
P K
PK
Fraziano
Maurizio
M
Colizzi
Vittorio
V
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015723:Gene Library; D016680:Genome, Bacterial; D005982:Glutathione Transferase; D006801:Humans; D016056:Immunodominant Epitopes; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods; D014376:Tuberculosis / Q000175:diagnosis*; D014611:Vaccination
D016056:Immunodominant Epitopes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase