Towards Improvement of Tuberculin Skin Test: Five Potential Antigenic Proteinselicited in vitro Specific Immune Reaction Against Mycobacterium tuberculous Species Using Sensitized Guinea Pig Models

摘要

Description of a new reagent of either single or multiple antigens to replace PPD remains challenging. Therefore, the current study attempted to fractionate culture filtrate of Mycobacterium bovis (M. bovis), using RF-HPLC. Obtained fractions were in vitro evaluated for their antigenicity by Lymphocytic Proliferation Assay (LPA) using PMBC from guinea pig models sensitized by heat killed M. bovis. Antigenic fractions were analyzed for its protein contents using SDS-PAGE. Multi-protein fractions were re-fractionated using shallower gradients of RF-HPLC. Obtained proteins were re-evaluated in vitro for their antigenic specificity by LPA and Gamma Interferon (γ-INF) using PMBC from sensitized guinea pigs by both Mycobacterium tuberculous (M. tuberculosis and M. bovis) and non-tuberculous Mycobacterium (M. intercellularae, M. avium, M. kansasi and M. fortuitum). The study revealed five proteins that elicited variable degrees of specific antigenicity only against tuberculous Mycobacterium sensitized PMBC. On SDS-PAGE analysis, selected proteins ranged between ~5 kDa up to ~25 KDa. Interestingly, negative skin reaction was revealed by all of the five selected proteins. Absence of pro-inflammatory factors, present in crude PPD from these pure proteinscould explain the in vivo failure of these in vitro provred antigenic proteins. The N-terminus sequencing of these proteins were carried out and the obtained sequences were searched for related Mycobacterium proteins using NCBI-protein blast. At this stage, the ORFs of the genes coding those proteins were characterized and currently, we are working on the cloning of these genes for mass production of corresponding proteins to be tried in different combinations with or without adjuvant.