Preparation Of Four Tuberculosis Latent Proteins And The Evaluation Of Their Immunological Characteristic

Objective: Aim to prepare the four recombinant proteins Rv2029c, Rv2659c, Rv2628andRv1813c and evaluate their immunological characteristic in Chinese populations.Methods:In this study, recombinant Rv2029c, Rv2659c, Rv2628and Rv1813c proteinswere cloned, expressed and purified by gene clone techniques. The expression conditionswere optimized. IFN-γ production from PBMC was assayed by ELISPOT in376casesconsisted of uninfected healthy subjects (103cases), subjects who were identified bystandard techniques as having latent tuberculosis infection (LTBI)(51cases), tuberculosispatients (116cases), subjects who received BCG vaccination (49cases) andnon-tuberculous respiratory disease patients (57cases). Serum latent antigen specific IgGlevel was assayed by ELISA in tuberculosis patients (62cases), LTBI subjects (36cases)and uninfected healthy subjects (72cases). To define a ROC curve, the SFC valuesstimulated by each antigen of LTBI subjects were taken as the positive group, and theactive TB patients were used as a control group. The performance of every antigen indifferentiation diagnosis of active infection and latent infection was evaluated.Results: The proteins with more than95%purity were obtained. When stimulated byrRv2029c, rRv2659c, rRv2628and rRv1813c, PBMC from LTBI subjects gave ELISPOTcounts that were significantly higher than those from TB patients and uninfected healthysubjects (p<0.05). When stimulated by rRv2029c, rRv2659c and rRv1813c, PBMC fromnon-tuberculosis respiratory disease patients gave ELISPOT counts that were significantlylower than LTBI subjects (p<0.05). The counts of Rv2628in non-tuberculous respiratorydisease group had no significant difference with latent infected subjects (p>0.05), also withuninfected healthy controls (p>0.05). The counts of rRv2029c, rRv2659c and Rv2628inBCG vaccinated positive conversion subjects had no significant difference with uninfectedhealthy subjects (p>0.05). The counts of rRv2029c, rRv2659c and Rv2628had no significant difference before and after BCG vaccination (p>0.05). The levels of serum IgGspecific for rRv1813c was significantly higher in TB patients than in LTBI subjects anduninfected healthy subjects (p<0.05). The levels of serum IgG specific for rRv2029c,rRv2659c, rRv2628were not significantly different between LTBI subjects and TBpatients (p>0.05). The area under the curve of rRv2029c was up to89.1%, higher thanrRv2659c (69.9%), rRv2628(68.8%) and rRv1813c (39%). The rRv2029c, rRv2659c,rRv2628stimulation gave detectable IFN-γ production in a higher proportion of personswith LTBI. rRv2029c had much higher sensitivity, whose positive rate in latent infectionwas more than80%, but also had a higher false positive rate in TB patients and uninfectedhealthy group. Although rRv2659c was less sensitive than rRv2029c, but its false positiverate was lowest in tuberculosis patients and uninfected healthy group. rRv2628hadconsiderable sensitivity with rRv2659c, and its false positive rate was higher thanrRv2659c, lower than rRv2029c.Conclusion:1. We successfully constructed four high expression strains which expresslatent tuberculosis associated proteins rRv2029c, rRv2659c, rRv2628and rRv1813c.Recombinant proteins with high purity were obtained.2. rRv2029c, rRv2659c, rRv2628and rRv1813c were be identified more easily by latent tuberculosis infected subjects andinduce stronger immune response by effector T cells compared with active tuberculosis.3.rRv2659c protein showed good performance in latent TB identification and active TBdifferentiation by ELISPOT technique. It is expected to become new diagnostic marker forlatent tuberculosis infection in Chinese population.4. rRv2029c, rRv2659c and rRv2628did not induce humoral immune response, but rRv1813c induced higher levels of specificIgG antibodies in active tuberculosis patients. rRv1813c may play a role in thepathogenesis of tuberculosis. In conclusion, our results provide experimental evidence forthe new diagnosis and vaccine markers of latent tuberculosis infection.