Mycobacterium Tuberculosis-specific Pe / Ppe Protein Used In The Preliminary Study Of Tb Serological Diagnosis

Human tuberculosis (TB) is a major infectious disease of the respiratory system. An early diagnosis followed by chemotherapy is the major control strategy. Improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement the diagnostic methods presently available.Proteins encoded by a 9.5-kb section of DNA called region of deletion 1 (RD1) of M. tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. The comparative genomics and bioinformatics have shown that in addition to RD1, 11 other genomic regions present in M. tuberculosis H37Rv are deleted in all strains of BCG. These regions cover 108 ORFs of M. tuberculosis H37Rv and are deleted from all 13 BCG substrains currently used as anti-TB vaccines in different parts of the world. A number of recent studies have demonstrated the diagnostic and vaccine potential of proteins encoded by RD1 and other RD regions. About 10% of the genome of M. tuberculosis codes for the PE and PPE families of proteins which are glycine rich and are exclusive to M. tuberculosis. The effects, which the PE/PPE family proteins — unique in their protein sequences and possible structure---may have on the immune system, have not been well documented. But recently, there are a few studies relevant to qualitative and quantitative the immune response of PE/PPE proteins in a clinical setting.The aim of the present study is to identify novel serological targets for use for the future serodiagnosis of tuberculosis. We evaluated the immunogenic properties of the PE/PPE family members, Rv3425, Rv3426, Rv3429, Rv2352c, Rv2353c, Rv3621c, Rv3622c, which are putative open reading frames conserved in M. tuberculosis but absent from the BCG, and compared them with other well-known antigens- the early secreted antigen target 6 (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv demonstrated that Rv3425, Rv3426,Rv3429, Rv2353c were expressed at the mRNA level, which indicated that they are functionally active genes and are expressed during exponential growth in vitro. We successfully cloned, expressed and purified the recombinant protein Rv3425, Rv3429, Rv2353c, CFP-10 and ESAT-6 in Escherichia coli, and these recombinant proteins were used to screen the sera of M. tuberculosis-infected patients, as well as those of BCG-vaccinated clinically healthy controls (n=28), by enzyme-linked immunosorbent assay (ELISA). The panel of patient sera comprised sera from pulmonary tuberculosis patients (n=50) and extra-pulmonary tuberculosis patients (n=32).It was shown that serum immunoglobulin G antibodies positive rate of recombinant protein antigen Rv3425, Rv3429, Rv2353c, CFP-10 and ESAT-6 were 70.0%, 22.0%, 16.0%, 78.0%, 36.0% in pulmonary tuberculosis patients and 59.4%, 18.8%, 12.5%, 65.6%, 34.4% in extra-pulmonary tuberculosis patients respectively, the specificity of these proteins were 100%, 96.4%, 96.4%, 96.4% and 100%. When the results of the three serodiagnostic tests of Rv3425, CFP-10, ESAT-6 were evaluated in combination, the sensitivity increased to 82% in pulmonary tuberculosis patients and to 68.8% in extra-pulmonary tuberculosis patients, the specificity was 96.4%.We also detected IgM antibody responses specific for recombinant protein antigen Rv3425, CFP-10 and ESAT-6, the sensitivity in pulmonary tuberculosis patients and extra-pulmonary tuberculosis patients were 32.0%, 74.0%, 78.0% and 29.0%, 64.5%, 61.3% respectively, the specificity of these proteins were 100%, 96.4%, 96.4%. When the results of the three serodiagnostic tests were evaluated in combination, the sensitivity increased to 88% in pulmonary tuberculosis patients and to 68.8% in extra-pulmonary tuberculosis patients, the specificity was 96.4%.From the study, we find that the recombinant M. tuberculosis Rv3425, CFP-10 and ESAT-6 proteins show statistically significant antigenic distinction between healthy BCG-vaccinated controls and TB patients (p<0.0001). We also find anti-IgG antibody response to recombinant Rv3425 is almost equal to CFP-10 and higher than ESAT-6. Our results highlight the immunosensitive and immunospecific nature of Mtb Rv3425 which is promising for use in the serodiagnosis of tuberculosis and has not beendescribed before.In conclusion, it is not possible to detect all of the antibodies against antigenic substances in the sera of all TB patients by using available serodiagnostic tests. However, the combined use of tests with three separate antigens maximizes the effectiveness of serodiagnosis.