Immunological Characterization Of Fusion Protein PstS1-LEP From Mycobacterium Tuberculosis

BackgroudTuberculosis (TB) is caused by Mycobacterium tuberculosis (M.tb).The treatment of Mtb is hindered by multi-and extensively drug-resistant M.tb, and human immunodeficiency virus (HIV) coinfection. Bacillus Calmette-Guerin (BCG) and antituberculosis drugs are used to prevent and chemotherapy TB, respectively. Thus, TB is also a serious public health issue in the world. TB control includes diagnosis, treatment, and prevention. With development of molecular technology, the specific or derivative antigens of M.tb are found to use as TB diagnosis and TB vaccine.ObjectivePstS1-LEP as a fusion protein was expressed in E.coli. IgG against PstSl-LEP were tested with ELISA in serum from pulmonary tuberculosis patients (PTB), extrapulmonary tuberculosis patients (EPTB), other pulmonary disease patients (PD) and healthy subjects (HCs) in order to evaluate the potential serodiagnosis of TB.MethodsTwelve Th line epitopes (15-mers), which obtained through screening20proteins of M.tb using online SYFPEITHI database, were connected end and end. Gene encoding LEP (LEP) using E.coli codon bias was obtained from DNA synthesis by Sangon Bioth and cloned into pET-28a-PstS1vector. The recombinant plasmids were transformed and expressed in E.coli BL21(DE3) with IPTG induction. The fusion protein (PstS1-LEP) was purified through anion exchange chromatography and renatured with combination dilution with dialysis. The western blot was used to detect antibodies in serum from TB patients and anti-PstS1monoclonal antibodies specific to PstS1-LEP. IgG antibodies against PstSl-LEP were detected using ELISA in serum from PTB, EPTB, and PD patients and HCs to evaluate the potential serodiagnostic value of PstSl-LEP.ResultsGene encoding LEP was obtained and cloned into pET-28a-PstS1vector. Gene sequencing confirmed that Recombinant plasmids have a concordance gene sequence with LEP’s. The PstS1-LEP protein was successfully expressed in E.coil. The concentration of PstS1-LEP was1.25mg/ml. PstSl-LEP showed positive reaction with the serum from PTB and anti-PstS1monoclonal antibody. The levels of Anti-PstS1-LEP IgG in PTB patients were significantly higher than in EPTB patients, PD patients, and HCs (P<0.05). HCs as control, ROC analysis showed that the cut off of ELISA was0.43. The sensitivity, specificity, PPV (positive predictive value) and NPV (negative predictive value) of PstS1-LEP was69.5%,68.4%,79.9%and55.3%. The sensitivity of PstS1-LEP in PTB patients was significantly higher than in EPTB patients (78.8%vs52.2%, x2=23.60, P=0.000). ROC curves using different controls obtained different cutoff. PD patients as control, the specificity of PstSl-LEP increased compared with HCs as control. The study showed that BCG vaccination affected serodiagnosis of PstSl-LEP, as the levels of IgG against PstSl-LEP in HCs with BCG scar was higher than in HCs without scar (P=0.008). Meanwhile, the levels of IgG against PstSl-LEP in HCs with PPD skin test-positive reactions were significantly higher than in HCs with PPD skin test-negative reactions (P<0.05), so, the size of skin test reaction may affect serodiagnosis of PstS1-LEP. This study showed that potential value of PstS1-LEP for TB serodiagnosis, but the sensitivity and specificity of PstS1-LEP did not meet the needs of clinical practice.ConclusionPstSl-LEP expressed in E.coli has immunogenicity, which provides a basis for further study on the value of PstS1-LEP as TB vaccine candidate.