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6-kDa early secretory antigenic target (ESAT6), a virulent factor of Mycobacterium tuberculosis, is involved in immune regulation. However, the underlying mechanism behind the activation and maturation of dendritic cells (DCs) by ESAT6 remains unclear. In this study, we investigated the effect on TLRs signaling on the regulation of ESAT6-induced activation and maturation of DCs. ESAT6 induced production of IL-6, TNF-α, and IL-12p40 in bone marrow-derived dendritic cells (BMDCs) from wild-type and TLR2-deficient mice, with this induction abolished in TLR4-deficient cells. NF-κB is essential for the ESAT6-induced production of the cytokines in BMDCs. TLR4 was also required for ESAT6-induced activation of NF-κB and MAPKs in BMDCs. ESAT6 additionally upregulated the expression of surface molecules CD80, CD86, and MHC-II, and also promoted the ability of CD4⁺ T cells to secrete IFN-γ via the TLR4-dependent pathway. Our findings suggest that TLR4 is critical in the activation and maturation of DCs in response to ESAT6.
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Animals , Mice , Cytokines , Dendritic Cells , Interleukin-12 Subunit p40 , Interleukin-6 , Mycobacterium tuberculosis , Mycobacterium , T-Lymphocytes , Toll-Like Receptor 4ABSTRACT
Objective To investigate the treatment evaluating value of ESAT－6 and CFP－10 in T－SPOT.TB kit for tuberculous vertebral osteomyelitis.Medthods This retrospective study analyzed 29 cases diagnosed as TVO in the First Affiliated Hospital of Kunming Medical University from January 2013 to January 2016. All cases were the first－time consultancy. The Wilcoxon－singed－rank－test and chi－square test were used to analyze the changes of ESAT－6 and CFP－10 in the procedure of treatment. The linear－regression analysis was used to analyze the relationship between ESR, CRP, VAS and two specific antigens.Results The pretherapeutic spot counts of ESAT－6 and CFP－10 were compared with the first and the last follow－up respectively. The results of two antigenic spots change showed a higher consistency (P＜0.05) .The positive rates of CFP－10 at the prior treatment, the first follow－up and the last follow－up were 86.20% , 79.31% and 58.62% respectively. The result of chi－square test showed a higher consistency (P＜0.05) . ESAT－6 only correlated with VAS. CFP－10 had the relationship with VAS and ESR. But all of these relativities were weak.Conclusion The decrease in the spot counts of ESAT－6 and CFP－10 suggest that the treatment is effective,and CFP－10 may be one available index to evaluate the treatment effect.
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Objective To investigate the effect of Shenmai injection on cytokines and ESAT-6/CFP-10 levels in patients with Chronic obstructive pulmonary disease(COPD)with pulmonary tuberculosis.Methods158 patients with COPD with pulmonary tuberculosis from the Department of Respiratory in our hospital were selected and divided into 2 groups, 79 cases in the control group treated with routine treatment, 79 cases in the experiment group treated with Shenmai injection on the basis of the control group, levels of neurotransmitters in the brain, serum ESAT-6 protein, CFP-10 protein, IFN-γ levels, cytokine levels, peripheral blood immune cell levels, and the clinical effect and the focus absorption efficiency were compared after the treatment.ResultsThe clinical total effective rate in the control group(84.81%)was lower than that in the experiment group(94.94%), with significant difference (P<0.05);the focus absorption efficiency in the control group(87.33%)was lower than that in the experiment group(96.21%), with significant difference (P<0.05);compared with the control group, serum ESAT-6 protein、 CFP-10 protein、IFN-γ levels were lower after treatment in the experiment group, the serum levels of TNF-α、IL-6、sIL-2R were lower, peripheral blood levels of NK cells, CD4+T lymphocytes and CD4+/CD8+ were higher, peripheral blood level of CD8+T lymphocytes was lower, with significant difference (P<0.05).ConclusionShenmai injection can significantly reduce the serum levels of TNF-α、IL-6、sIL-2R and ESAT-6/CFP-10 in patients with COPD with pulmonary tuberculosis, improve cellular immune function, promote the absorption of lesions effectively,and improve clinical efficacy.
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Objective To investigate the Mycobacterium tuberculosis infections in 2014 among Beijing army recruits, and evaluate a new method for screening latent tuberculosis infections.Methods A total of 194 army recruits were subjected to chest X-ray examination purified protein derivative(PPD) skin test, antibody detection, and interferon gamma release assay(IGRA) by ELISA combined with recombinant protein CFP10-ESAT6 and latent infection protein Rv2628.Results The positive rates of PPD skin test and antibody test were 49.7% and 15.5%, respectively.The latent infection rate of IGRA test was 22.2% in 194 cases after CFP10-ESAT6 stimulation.After stimulation of latent tuberculosis infection(LTBI) with Rv2628, IFN-γ level was significantly higher than that in healthy control group (P0.05).There was no significant difference between antibody negative and positive groups after stimulation by CFP10-ESAT6 and Rv2628 (P>0.05).The area under the ROC curve of Rv2628 diagnosis of tuberculosis infection was 0.84.When Youden index was 0.621,the specificity was 94.7% and sensitivity was 67.4%.ConclusionCombined detection of antigens Rv2628 and CFP10-ESAT6 specific IFN-γ values can be potentially used for differential diagnosis of active or latent tuberculosis infections.
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Objective To investigate the effects of Mycobacterium tuberculosis heparin-binding he-magglutinin (HBHA) on the polarization of mouse bone marrow-derived macrophages (BMDM) and a mu-rine macrophage cell line(Raw264.7 cells). Methods After HBHA was confirmed to be able to enter into macrophages by immunofluorescence, mouse BMDM and Raw264. 7 cells were treated with LPS (100 ng/ml)+IFN-γ (2.5 ng/ml),IL-4 (20 ng/ml),HBHA (1 μg/ml,3 μg/ml,6 μg/ml,10 μg/ml) and ESAT-6 (5 μg/ml),respectively. IL-6,IL-12 and TNF-α in the supernatants of cell culture were measured by ELISA. RT-PCR was performed to detect the expression of molecular markers of macrophage polarization [inducible nitric oxide synthase (iNOS), TNF-α, Arg-1 and CD206] at mRNA level. Western blot assay was used to detect the expression of iNOS and Arg-1 at protein level. Results Increased secretion of IL-6, IL-12 and TNF-α in the supernatants of cell culture and enhanced expression of iNOS and TNF-α at mRNA level were observed after treating BMDM with Mycobacterium tuberculosis HBHA. Similarly,in HBHA-trea-ted Raw264.7 cells,the secretion of IL-6 and the expression of TNF-α were also up-regulated. Mycobacteri-um tuberculosis HBHA and ESAT-6 had similar effects on murine macrophages. Neither of them could in-crease the expression of Arg-1,a molecular marker of M2 macrophages,in BMDM at protein level,but both enhanced the expression of iNOS,a molecular marker of M1 macrophages,in BMDM and Raw264.7 cells. Conclusion Mycobacterium tuberculosis HBHA can induce the polarization of murine macrophages to M1 phenotype.
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Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.
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Objective To observe the expression of immune substances secreted by peripheral bloodγδT cells after stimulated by early secreted antigenic target-6(ESAT-6),and explore its mechanism of signaling pathway.Methods Peripheral blood mononucle-ar cells(PBMC)were collected from health human blood with the ficoll density gradient centrifugation method,and theγδT cells were separated from the PBMC with flow cytometry;the inhibitor of TLR-4 signaling pathway(E5564)was used to cocultured withγδT cells to inhibit the function of TLR-4,and the change of TLR-4 was analyzed by the methods of PCR and Western blot.ESAT-6 were used to stimulate theγδT cells,and the control group without any stimulating factor was established,then the expression levels of IL-17,TNF-α,IFN-γwere determined by ELISA method after 0、1、3、6、9 and 12 days.Results The results of PCR and Western blot showed that ESAT-6 could increase the expression of TLR-4(P<0.01);The results of ELISA showed that ESAT-6 could enhance the expression of IL-17,TNF-αand IFN-γ(P<0.01),and the inhibitor of TLR-4(E5564)could decrease the expres-sion of IL-17,INF-α,IFN-γ(P<0.01).Conclusion ESAT-6 can induceγδT cells to produce more IL-17,TNF-α,IFN-γ,and the mechanism of which maybe concerned with TLR-4 signaling pathway.
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This study aimed to construct a bicistronic DNA vaccine expressing fusion antigen Hsp65-Esat-6 of Mycobacterium tuberculosis with cytokine GM-CSF as a molecular adjuvant (pIRES-Hsp65-ESAT-6-GM-CSF, pIRHEG), and the immune response in mice. C57BL/6 mice were immunized with the recombinant plasmid to detect the titer of antibodies, lymphocyte proliferation, the ratio of CD4+, CD8+T cell and IFN ~ γï»IL-2 secretion. The titer of antibody, lymphocyte proliferation, the ratio of CD4+T and CD8+T cells and IFN ~ γ, IL-2 secretion of pIRHEG group was significant higher than other recombinant plasmid groups, which significant differed by statistical mean. The bicistronic DNA vaccine could induce an effective immune response in mice and could be used as vital ingredient of a new tuberculosis vaccine candidate.
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The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Antigens, Bacterial , Mycobacterium tuberculosis/immunology , Peptides , Tuberculosis/diagnosis , Antigens, Bacterial/immunology , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Peptides/immunology , Sensitivity and Specificity , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/immunology , Tuberculosis/immunologyABSTRACT
Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.
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The aim of this work was to study the difference in interferon gamma (IFN-gamma) production by T lymphocytes after early secretory antigen target 6 (ESAT-6) or purified protein derivate (PPD) stimulation in whole blood culture supernatants from children with suspected tuberculosis (TB) disease (n = 21), latent TB infection (n = 16) and negative controls (NC) (n = 22) from an endemic area in Brazil. The concentration of IFN-gamma (pg/ml) was measured by enzyme linked immunosorbent assay and the differences in the IFN-gamma levels for each group were compared and evaluated using an unpaired Student's t-test; p values < 0.05 were considered significant. Measurement of IFN-gamma levels after ESAT-6 stimulation raised the possibility of early diagnosis in the latent TB group (p = 0.0030). Nevertheless, the same group showed similar responses to the NC group (p > 0.05) after PPD stimulation. The IFN-gamma assay using ESAT-6 as an antigenic stimulus has the potential to be used as a tool for the immunodiagnosis of early TB in children.
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Adolescent , Child , Child, Preschool , Female , Humans , Male , Antigens, Bacterial , Bacterial Proteins , Interferon-gamma/biosynthesis , Mycobacterium tuberculosis/immunology , Tuberculosis, Pulmonary/diagnosis , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Sensitivity and Specificity , Tuberculosis, Pulmonary/immunologyABSTRACT
Objective To sensitize the T-cell in the peripheral blood of the active tuberculosis pa-tients by rCFP-10/ESAT-6 fusion protein, phytohaemaggiutinin(PHA) and physiologic saline, and to detect the IFN-γ to approach the significance of the tuberculosis infection. Methods One hundred and eleven pa-tients were diagnosed by clinical definite, 292 undergraduate students were chosen by X-ray and PPD-selec-fion as volunteers. 3.0 ml of blood was taken from each volunteer, rCFP-IO/ESAT-6, PHA and physiologic saline were added into each 1.0 ml, respectively. The A valule and antibody of IFN-γ were assayed by ELISA. Results Treated with rCFP-10/ESAT-6 group: the A value average of patients group was 1. 3885±0.6236, students group was 0.2944±0.0917. Intergroup t'=16.4259, P<0.05, set>0.42 as cut-off, the positive rate of patients group was 93.58%, students group was 13.07%. Treated with PHA group: the A value average of patients group was 1.2463±0.5541, of which the other was 0.5613±0.064, t'=19.1797,P<0.05. Treated with physiologic saline group:the A value average of patients group was 0.0772±0.0444,of which the other was 0.0290±0.0235,t'=13.9487,P<0.05. All had significant deviation. The antibody positive rate of the patients group was 66.36%, the students group was 7.19%. Conclusion rCFP-10/ESAT-6 as specific antigen, the sensitivity of IFN-γ release assay by ELISA is above 90%. No matter specific or non-specific disposal, the active tuberculosis patients have higher IFN-γ, release level and antibody than the control group.
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A tuberculose continua sendo um grave problema social e de saúde, afetando milhões de pessoas anualmente. A vacina Bacille Calmette-Guerin (BCG), usada no controle profilático, é incapaz de conter a progressão da doença, que usualmente se manifesta através da queda da imunidade celular do indivíduo. O diagnóstico da tuberculose em seus estágios iniciais, aliado à poliquimioterapia, pode contribuir para o controle da disseminação da infecção. Os atuais métodos de diagnóstico apresentam problemas, como: baixa sensibilidade da baciloscopia; longo tempo de realização das culturas microbiológicas; e baixa especificidade do teste cutâneo com o derivado protéico purificado do M. tuberculosis. Novos métodos de diagnóstico que utilizam antígenos específicos (por exemplo, os conhecidos em inglês como o early secreted antigenic target 6-kDa e o culture filtrate protein 10-kDa), estão sendo testados. Os genes que codificam esses antígenos estão localizados na região de diferença 1 do M. tuberculosis, M. africanum e M. bovis, mas estão ausentes no M. bovis (BCG) e na maioria das micobactérias do meio ambiente. Métodos de diagnóstico baseados na produção de interferon-gama por linfócitos T, em resposta a esses antígenos, como o QuantiFERON-TB® e o T SPOT.TB®, estão sendo testados, e superam o teste cutâneo com o derivado protéico purificado nas seguintes características: maior sensibilidade; menor reatividade cruzada devido à vacinação com o BCG ou infecção por micobactérias do meio ambiente; e tempo de execução. A introdução de métodos de diagnóstico mais específicos e sensíveis, assim como um maior entendimento dos mecanismos moleculares e celulares que regulam a interação parasito-hospedeiro, pode contribuir para um eficiente combate à tuberculose.
Tuberculosis remains a serious social and public health problem, affecting millions of people annually. The bacille Calmette-Guerin (BCG) vaccine, used prophylactically, does not impede the progression of the disease, which usually manifests as decreased cellular immunity. Early diagnosis, together with polychemotherapy, can control the dissemination of the tuberculosis infection. The current diagnostic methods present certain problems. Such problems include the low sensitivity of sputum smear microscopy, the fact that performing microbiological cultures is quite time-consuming, and the low specificity of the skin test with the purified protein derivative of M. tuberculosis. New diagnostic methods, which use specific antigens such as the early secreted antigenic target 6-kDa and culture filtrate protein 10kDa, are being evaluated. The genes that encode these antigens are located in the DNA region of difference 1 of M. tuberculosis, M. africanum and M. bovis. However, they are absent from the M. bovis (BCG) and from most environmental mycobacteria. Diagnostic methods such as QuantiFERON-TB® and T SPOT.TB®, which are based on the production of interferon-gamma by T lymphocytes, in response to those antigens, are being tested and have been found to outstrip the purified protein derivative skin test in the following characteristics: greater sensitivity; lower cross-reactivity due to BCG vaccination or infection with environmental mycobacteria; and execution time. The introduction of diagnostic methods that are more specific and sensitive, together with gaining a better understanding of the molecular and cellular mechanisms that regulate the parasite-host interaction, can increase the efficiency of strategies devised to combat tuberculosis.
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Humans , Mycobacterium tuberculosis/immunology , Serologic Tests/methods , Tuberculosis/diagnosis , Antigens, Bacterial/immunology , Immunity, Cellular , Immunocompetence/immunology , Immunocompromised Host/immunology , Sensitivity and Specificity , Tuberculosis/immunology , Tuberculosis/prevention & controlABSTRACT
The RD-1 locus has been considered crucial in the pathogenesis of M.tuberculosis, the RD-1 locus is 9.5 kb and spanning open reading frames Rv3871 to Rv3879c encoding 9 different proteins separately.The RD-1 locus is missing in all bacillus Calmette-Guerin(BCG) strains, and is one of the key virulence factor in M.tuberculosis.The RD-1 locus participates in a new secreting system named ESX-1, which can facilitate the secretion of some special proteins.The two important proteins encoded by the RD-1 locus named CFP-10 and ESAT-6 can form a tight 1∶1 complex, and has been shown to be coordinately secreted and lead to a strong T cell response, which suggests that these two proteins may act as ideal target antigens in diagnosis and prevention of tuberculosis(TB).
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Objective To obtain sufficient recombinant multiepitope peptide of ESAT-6.Methods DNA encoding ESAT-6 peptide was amplified by PCR using a pair of primers which were designed in accordance with the reported ESAT-6 gene sequence.The ESAT-6P gene was inserted into the prokaryotic expression vector pET-30a with the N-terminal 6His-tag.The recombinant peptide vector pET30a-ESAT-6P was transformed into E.coli BL21(DE3) via chemical transformation.The positive clone was screened by means of PCR.The target peptide was expressed in E.coli after induction with IPTG and analyzed by SDS-PAGE.The immunogenicity of the peptide was evaluated by dot immunobinding assay.Results The target peptide was successfully expressed and purified.The solubility analysis showed that the recombinant peptide existed as inclusion body in the E.coli.DIBA indicated that the ESAT-6P was specifically reactive to sera from TB patients.Conclusions The recombinant multiepitope peptide of ESAT-6 engineering bacteria has been obtained,which will be quite helpful to develop novel specific diagnostic reagents and effective vaccine.
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BACKGROUND:The tuberculin skin test, which has been used for years for the diagnosis of latent tuberculosis, has many limitations, including false-positive results in individuals who were vaccinated with BCG. We evaluated the usefulness of a recently developed interferon-gamma assay (QuantiFERON-TB Gold) in the diagnosis of latent tuberculosis. METHODS:We performed the QuantiFERON-TB Gold assay in the following groups: 1) individuals with negative responses of tuberculin skin test in the regular health checkups for two consecutive years (n = 14), 2) individuals with no abnormal findings in low dose computed tomography in a health checkup (n = 22), 3) individuals with stable tuberculosis in low dose computed tomography in a health checkup (n = 10), 4) patients with M. tuberculosis in culture (n = 23), 5) patients with nontuberculous mycobacteria in culture (n = 6). RESULTS:In the QuantiFERON-TB Gold assay, all the group 1 showed negative results. 65.2% of the group 4 showed positive QuantiFERON-TB Gold results, while all the group 5 showed negative results. 22.7% of the group 2 and 60.0% of the group 3 showed positive QuantiFERON-TB Gold results. In addition, it was revealed that the stimulation with CFP-10 played a major role in the induction of interferon-gamma secretion. CONCLUSION:The QuantiFERON-TB Gold assay shows promise for the immunodiagnosis of latent tuberculosis using a whole-blood.
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Humans , Diagnosis , Immunologic Tests , Interferon-gamma , Latent Tuberculosis , Mycobacterium bovis , Nontuberculous Mycobacteria , Skin Tests , Tuberculin , TuberculosisABSTRACT
Objective To construct the eukaryotic expression plasmids of Mycobacterium tuberculosis ESAT-6 antigen and to investigate its immunogenicity in mice. Methods The sequence encoding ESAT-6 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA. Then, the amplified fragment was sub-cloned into pVAC expression vector. The eukaryotic expression plasmid pVAC-ESAT-6 was transfected into Vero E6 cells with liposome. The mRNA expression of pVAC-esat-6 in Vero E6 cells was detected by RT-PCR and its expression product was analyzed by Western-blot. The plasmid pVAC-esat-6 was introduced into the mice by gene gun injection. The specific antibody to ESAT-6 and the level of IFN-? in supernatant of spleno-lymphocyte cultures were detected by ELISA methods. Results The recombinant plasmid pVAC-esat-6 was successfully constructed, and the expression of ESAT-6 was also detected in vitro. After vaccinated three times, the mice produced specific antibody and the level of IFN-? in supernatant of spleno-lymphocyte cultures in group of pVAC-esat-6 was significantly higher than group of vector alone (P
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Objective To clone and express the gene encoding ESAT antigen of Mycobacterium tuberculosis (chinese stain). Methods Genome of mycobacterium tuberculosis was extracted. Then, the full length cDNA encoding ESAT protein was amplified by PCR and cloned into prokaryotic expressing vector pGEX5T and sequenced. pGEX5T ESAT was expressed in k802 Ecoli, and the expressed protein was determined by western blot using the sera from ten patients with tuberculosis. Results One specific band of 0.3kb or so was obtained and sequencing result was identical to that reported from Genbank. The expressed protein could be specifically recognized by the sera from tuberculosis patients. Conclusions The full length cDNA of Mycobacterium tuberculosis (Chinese strain) ESAT protein was cloned and expressed successfully, which will be helpful in further studies on diagnosis and treatment of tuberculosis.
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To construct expressing vector carrying esat6 gene and express this protein in Streptococcus gor-donii GP251. esat6 gene was amplified by PCR with specific primer from genome of Mycobacterium tuber-culosis (MTB)H37Rv. Inserted esat6 into the pMD18-T vector by T/A clone to get recombinant vector pMD18-esat6. Then digested pMD18-esat6 with restriction enzyme, esat6 was cloned to vector PSMB104 and expressed in Streptococcus gordonii GP251. The expression of esat6 protein was detected by Tricine-SDS-PAGE and Western-blot, ELISA technique was also used to detect its secretory volume. Re-striction endonuclease, PCR, Tric ine-SDS-PAGE and Western-blot confirmed that esat6 gene was cloned into expressing vector successfully, and a 10 kD protein secreted in Streptococcus gordonii GP251, this pro-tein has a good immunogenicity. The expression vector of esat6 gene was constructed, and esat6 protein ex-pressed in Streptococcus gordonii1 successfully, it will be benefit for future study.
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Objective:To construct prokaryotic expression vector carrying esat6 gene and express in E. coli. Methods: The MTb esat6 gene was amplified by PCR,then cloned into pQE30 plasmid, sequenced and then cloned into pET32a( + ) plasmid. Thus two kinds of prokaryotic expression vectors were constructed. Results: After being transformed into the E. coli and inducted with 1mM IPTG,no protein was expressed in the pQE30 - ESAT6 system, but a recombinant protein, about 19 kDa,was expressed in the pET32a( + ) - ESAT6 system. In the presence of 1mM IPTG for 4h,the protein was expressed to the maximum. The protein existed in cytoplasm in soluble form and represented 42% total protein of E. coli. It's antigenicity was confirmed by Westem blotting. The protein was purified through the Ni - NTA resin and the purity reached 92 % . Conclusion : The prokaryotic expression vector (pET32a( + ) - ESAT6) was constructed successfully,and the rESAT6 was obtained,providing an experimental basis for application of rESAT6.