Isolation and characterization of a 31 kDa mycobacterial antigen from tuberculous sera and its identification with in vitro released culture filtrate antigen of mtb H37Ra bacilli

Antigens released in vivo are of considerable interest in the immunodiagnosis of infectious diseases. Circulating antigen was isolated from bacteriologically confirmed tuberculous sera by ammonium sulphate precipitation. The protein fraction between 36%, and 75%, ammonium sulphate was reactive with tuberculosis (TB) sera showing the presence of circulating tubercular antigen (CTA). Fractionation of CTA on ultrogel AcA 34 gel filtration column gave 3 protein fractions CTA1, CTA2 and CTA3. CTA2 showed maximum antigenic activity by sandwich enzyme-linked immunosorbent assay (ELISA). SDS-PAGE fractionation and seroreactivity studies showed the presence of highly reactive tubercular antigen in CTA2-7 protein fraction by sandwich ELISA. Further fractionation of CTA2-7 on cation exchange fast-protein liquid chromatography (FPLC) gave 4 antigenic fractions, of which CTA2-7D was seroreactive similar to 31 kDa antigen (ESAS-7F) isolated from in vitro culture medium. Furthermore, CTA2-7D could inhibit binding of in vitro released ESAS-7F to affinity purified antibodies in inhibition ELISA. CTA2-7D antigen may be used as a target antigen in confirming active tubercular infection. Biochemical characterization showed circulating antigen CTA2-7D to be a lipoglycoprotein is released in vivo. ESAS-7F as a glycoprotein is released in vitro culture.     

Sero-reactivity of two different antigenic protein fractions of M.tb H37Ra excretory-secretory antigen in pulmonary and extra-pulmonary tuberculosis

Excretory-secretory proteins of Mycobacterium tuberculosis H37Ra, have been of diagnostic interest in pulmonary (PTB) and extrapulmonary tuberculosis (EPTB). Two different excretory-secretory antigenic proteins of M.tbH37Ra viz., EST-DE1 (a 6% TCA soluble and DEAE anion exchange purified antigen) and ESAS-7 (50% ammonium sulphate solubilized and SDS-PAGE fractionated antigen) were studied in stick-indirect penicillinase ELISA for detecting tuberculous IgG antibodies in serum samples of pulmonary as well as extrapulmonary tuberculosis (tuberculous lymphadenopathy (TBLN), tuberculous meningitis (TBM), bone & joint tuberculosis (B&J TB), abdominal tuberculosis (Abd. TB) patients. The ESAS-7 antigen has shown comparatively better seroreactivity (90%) than that of EST-DE1 antigen in pulmonary tuberculosis cases. The overall specificity of 93.2% using ESAS-7 antigen was also found better compared to 86.4% obtained using EST-DE1 antigen. Further, in extra pulmonary tuberculosis group, using ESAS-7 antigen 84% (21/25) of histopathologically confirmed TBLN cases and 90% (9/10) clinically diagnosed and ATT responded TBM cases showed positive reaction for tuberculous IgG antibody. The per cent positivity using EST-DE1 antigen was however comparatively low in TBLN and TBM cases, (76% and 80% respectively). In histopathologically proven bone and joint tuberculosis and abdominal tuberculosis cases EST-DE1 antigen showed better sensitivity of 75% and 83.3% respectively in IgG antibody detection compared to that of ESAS-7 antigen (50% and 66% respectively). From the present study, it can be envisaged that ESAS-7 antigenic fraction has a good potential in the diagnosis of pulmonary and certain extra-pulmonary tuberculosis infection (TBLN & TBM) whereas EST-DE1 was found to be better in detecting specific antibodies in bone & joint and abdominal tuberculosis.     

Detection of free and immune-complexed serine protease and its antibody in TB patients with and without HIV co-infection.

OBJECTIVE: To understand the usefulness of detecting tuberculous IgG antibodies against mycobacterial excretory-secretory 31 kDa serine protease antigen (SEVA TB ES-31) and circulating free and circulating immune-complexed (CIC) serine protease in TB patients with and without HIV infection. DESIGN: Serum was collected from 144 individuals: patients with TB, with TB-HIV co-infection and HIV infection only, and ill and healthy controls. SEVA TB ES-31 antigen, a serine protease isolated from Mycobacterium tuberculosis H37Ra culture fluid, was used in indirect penicillinase ELISA to detect tuberculous antibodies. Similarly, affinity purified anti-ES-31 antibody was used in sandwich ELISA to detect circulating free and CIC serine protease. RESULTS: There was less sensitivity for tuberculous antibody in HIV-infected TB patients (46%) than in those with TB alone (87%) using mycobacterial serine protease. However, the sensitivity of detection of TB in the presence of HIV increased to 87% by concomitant detection of circulating free and CIC serine protease antigen. CONCLUSION: Detection of free and CIC tuberculous serine protease antigen along with antibody is more useful for detecting TB in the presence of HIV co-infection.     

Growth phase-associated changes in protein expression in Mycobacterium smegmatis identify a new low molecular weight heat shock protein

De novo protein synthesis and the heat-shock response during different stages of bacterial culture of Mycobacterium smegmatis LR222 were investigated. A discontinuance in the increase in number of colony forming units occurred at mid-exponential phase of growth. This coincided with a plateau in the ATP content of the culture, a reduction in the synthesis of exponential phase proteins (58, 30.5, and 20 kDa), a transitory synthesis of a 32 kDa protein and the induction of stationary-phase proteins (48, 46, 31, 25, and 20 kDa). The response to heat shock showed a growth-phase dependency, with the highest fold-induction of the 75 kDa (DnaK) protein occurring during the transitory cessation in the increase in CFU, while the greatest increase of the 95 kDa, 66 kDa (GroEL), and approximately 17 kDa (a doublet) proteins occurred during stationary phase. The approximately 17 kDa doublet was resolved into four polypeptides by two-dimensional electrophoresis. Mass spectrometric analysis of the sequence of one polypeptide (named Hsp17-2, 16.8 kDa) revealed significant homology to a conserved, 16.2 kDa, hypothetical protein of unknown function in Mycobacterium tuberculosis H37Rv. The increased synthesis of Hsp17-2 in response to heat shock suggests that it may represent a new low molecular weight heat shock protein.     

Isolation and evaluation of diagnostic value of two major secreted proteins of Mycobacterium tuberculosis

Two secreted antigens of Mycobacterium tuberculosis, namely the antigen 85 complex (30/31) and 38kDa antigens, were purified from the whole culture filtrate by using two dimensional preparative electrophoresis and anion exchange chromatography, respectively. Individual components of the antigen 85 complex namely, antigen 85A, 85B and 85C, were separated using hydrophobic interaction chromatography. The humoral antibody activity to these antigens in sputum positive cases of active pulmonary tuberculosis and normal healthy volunteers was determined by enzyme linked immunosorbent assay (ELISA) and immunoblot. Recombinant 38kDa and antigen 6 were used as reference antigens for the assay. None of the healthy volunteers reacted with the 38kDa antigen, while 52% of the TB sera reacted with it. Of the three components of the antigen 85 complex, 85B gave the highest positivity of 40 per cent. The results of combination of 38kDa with antigen 6 offered better results with 76% positivity.     

结核杆菌Ag85A及ESAT-6双价抗原融合表达载体的构建及其在大肠杆菌中的高效表达

目的　为结核病新型疫苗研究提供靶基因和靶抗原。方法　采用PCR扩增的方法获得结核杆菌两种免疫保护性抗原Ag85A及ESAT - 6的基因 ,将其定向克隆入真核及原核穿梭表达型载体 pBK -CMV构建含嵌合目的基因的重组质粒 ,转化大肠杆菌后用IPTG进行诱导表达 ,并通过SDS -PAGE和Western -blotting对表达蛋白进行初步分析。结果　 1)从结核杆菌H37Rv株基因组DNA中扩增出Ag85A及ESAT - 6基因。 2 )成功构建了结核杆菌Ag85A及ESAT - 6双价抗原融合表达质粒 pBK - 85A -E6。 3)重组质粒 pBK - 85A -E6经IPTG诱导后能在大肠杆菌中稳定表达 38kDa的融合蛋白。 结论　成功构建了结核杆菌Ag85A及ESAT - 6双价抗原融合表达载体 ,并在大肠杆菌中实现了稳定表达 ,为进一步研究其在结核病基因工程疫苗研制中的应用奠定了基础。     

结核分枝杆菌重组Mtb8.1蛋白自诱导表达与抗原性分析

目的 通过自诱导表达系统重组表达结核分枝杆菌H37Rv菌株可溶性蛋白抗原Mtb8.1, 并对其抗原性进行分析。方法 应用PCR技术扩增结核分枝杆菌H37Rv 菌株Mtb81蛋白抗原编码序列, 将其插入克隆载体pMD18-T, 酶切后插入表达载体PET28a, 通过优化培养基和发酵工艺, 摸索出一条高效的可溶表达方法, 以间接ELISA法研究其免疫学特性。结果与结论 重组的Mtb81高效可溶性表达, 纯化后纯度达95%以上;用纯化的重组蛋白抗原作为包被抗原建立的间接ELISA法检测200份血清样本, 阳性率达34.0%, 阴性率达91.0%, , 符合率达62.5%。     

Serological response (Western blot) to fractions of Mycobacterium tuberculosis sonicate antigen in tuberculosis patients and contacts.

OBJECTIVE: To assess the serological response to fractions of Mycobacterium tuberculosis sonicate antigen by Western blot analysis in patients with tuberculosis and contacts. METHODS: We studied 71 individuals including 43 patients with active tuberculosis, 16 contacts and 12 healthy blood donors. For Western blot analysis, M. tuberculosis (H37Rv strain) sonicate antigen extract was fractionated by electrophoresis on polyacrylamide gel (SDS-PAGE). RESULTS: We obtained antibody responses directed against four antigenic fractions with molecular weights of 71, 65, 26-38 and 19 kDa. Sixty per cent of pleural tuberculosis and 52.4% of smear-positive pulmonary tuberculosis had whole responses against all four fractions; there were no partial responses in these groups. For patients with smear-negative pulmonary tuberculosis whole responses were 17.6% and partial responses 41.2%. All contacts whose tuberculin tests converted from negative to positive (three cases) reacted exclusively against the 19 kDa fraction. CONCLUSIONS: Western blot-positive results in patients with pleural and smear-positive pulmonary tuberculosis were characterised by a whole pattern against all four antigenic fractions, whereas patients with smear-negative pulmonary tuberculosis showed heterogeneous results. The exclusive response against the 19 kDa fraction observed in contacts with tuberculin conversion could help to identify candidates for preventive therapy.     

结核分枝杆菌14000抗原基因的克隆、表达纯化及抗原性分析

目的对结核分枝杆菌14 000抗原基因进行克隆表达及纯化,并评价其抗原性。方法以结核杆菌H37Rv株基因组DNA为模板,用PCR方法获得目的基因构建大肠杆菌高效表达株;亲和层析柱纯化重组蛋白;进SDS-PAGE电泳鉴定;通用ELISA法进行重组蛋白的抗原性检测分析。结果获得了结核分枝杆菌抗原14 kD基因,在大肠杆菌BL21中高效表达,Western印迹结果证实该重组蛋白能与抗结核分枝杆菌多克隆抗体发生特异免疫结合反应。ELISA分析中,该纯化的重组抗原敏感性、特异性和准确性分别为:52.5%、96.7%和67.2%,具有良好的抗原性和较高的特异性。结论14 kD重组蛋白抗原具备良好的抗原性与特异性,为结核病诊断、重组疫苗应用和免疫效应检测以及抗原、抗体的大规模制备打下基础。     

Serological and immunochemical properties of carbohydrate-containing fraction from mycobacterium tuberculosis H37Rv

Using affinity chromatography on concanavalin A (Con A) sepharose CL 6B, a carbohydrate-containing fraction was derived from Mycobacterium tuberculosis H37Rv sonicate. Surprisingly, the main component of Con A fraction was a protein having a molecular weight of a 30-kD range which is generally absent in the Con A-adsorbed fraction from the culture filtrate. ELISA by means of an antimycobacterial monoclonal antibody panel showed that the 30-kD range of Con A fraction contained antigen 85. It is suggested that the derived components antigen 85 are a glycosylated form of the proteins associated with the mycobacterial cell wall. The Con A fraction, antigen 85 (Department of Virology, Pasteur Institute, Brussels, Belgium), and PPD (Batch RT 45, Stattens Seruminstitute, Denmark) were used for ELISA determination of antimycobacterial antibody titers in the sera of 30 patients with pulmonary tuberculosis, 28 patients with nonspecific lung diseases (bronchitis and/or asthma, pneumonia), as well as in the sera of 12 healthy volunteers. The sensitivities were 46.42, 57.34, and 72.33% and the specificities were 36.97, 26.73, and 56.75% for Con A fraction, antigen 85, and PPD, respectively. The authors suggest that serodiagnostic properties of Con A fraction are extremely limited.     

结核分支杆菌38 000蛋白质抗原在大肠杆菌中高效表达

目的通过结核分支杆菌３８０００蛋白质抗原编码基因在大肠杆菌中稳定表达,以获得大量纯化的３８０００蛋白质抗原,进而研究其免疫学特性。方法采用ＤＮＡ重组技术构建结核分支杆菌３８０００蛋白质抗原表达载体,双酶切和聚合酶链反应（ＰＣＲ）鉴定转化子,阳性重组子转化大肠杆菌,并诱导表达外源蛋白;十二烷基磺酸钠聚丙烯酰胺凝胶电泳（ＳＤＳＰＡＧＥ）和免疫印迹法鉴定３８０００蛋白质抗原在大肠杆菌中的表达;对染色的凝胶扫描,以测定目的蛋白质表达水平。诱导的工程菌抽提包涵体,以确定３８０００蛋白质抗原在大肠杆菌中的表达形式。结果菌体蛋白经ＳＤＳＰＡＧＥ,含阳性重组质粒的菌体蛋白中出现一条新蛋白带,表达量占菌体总蛋白的３６％～４０％。肺结核患者的血清和结核分支杆菌免疫的羊血清与重组３８０００蛋白质均呈阳性反应。３８０００蛋白质抗原在大肠杆菌中表达方式主要以包涵体形式。结论构建的大肠杆菌重组体能高效表达结核分支杆菌３８０００蛋白质抗原,包涵体的表达形式有利于蛋白质纯化和蛋白质稳定,蛋白质必须经过正确折叠才具有生物学活性     

Isolation and characterisation of in vivo released 41 kDa mycobacterial antigen in pulmonary and bone and joint tuberculosis and its identification with H37Ra in vitro released antigen.

OBJECTIVE: To isolate and characterise in vivo released 41 kDa mycobacterial antigen in pulmonary and bone and joint tuberculosis (TB) and its identification with in vitro released ES-41 kDa antigen. DESIGN: Circulating antigen was isolated from confirmed pulmonary tuberculosis serum (PTS) and bone and joint tuberculosis serum (BJS) by trichloroacetic acid precipitation and further fractionation by fast-protein liquid chromatography (FPLC). RESULTS: Fractionation of PTS and BJS by gel filtration column gave six protein fractions each. PTS-G3 and BJS-G3 showed maximum antigenic activity with ELISA. Further fractionation of PTS-G3 and BJS-G3 on cation exchange FPLC gave four different fractions each, of which BJS-G3B was seroreactive similarly to in vitro released 41 kDa antigen (ES-41) isolated from culture medium, whereas PTS-G3C was slightly less seroreactive. BJS-G3B could inhibit binding of in vitro released ES-41 to affinity purified antibodies in inhibition ELISA at lower concentrations than PTS-G3C (2 vs. 20 ng/ml), showing the identical nature of the antigens. Biochemical characterisation showed that circulating antigen PTS-G3C, BJS-G3B and in vitro released ES-41 antigen were lipoproteins in nature. CONCLUSION: This study helped to demonstrate the presence of 41 kDa antigen in the serum of pulmonary and bone and joint TB patients and its identification with H37Ra in vitro released 41 kDa antigen.     

Isolation, characterization, and specificity of a glycoprotein antigen from Mycobacterium kansasii

We have isolated in highly purified form and characterized a glycoprotein antigen from culture filtrates of Mycobacterium kansasii. Immunoelectrophoretic studies demonstrated that this antigen is present only in M. kansasii among 14 species of mycobacteria studied. It is a potent tuberculin skin test antigen, but skin test reactions to it in sensitized guinea pigs display only limited specificity. Enzyme-linked immunoabsorbent antibody assays of serum samples from patients suffering from diseases caused by M. kansasii, M. intracellulare, and M. tuberculosis display no specificity with this antigen. These findings are best explained by hypothesizing a single highly antigenic determinant that is present as a moiety on many mycobacterial antigens and is widely shared among mycobacteria.     

Identification and preliminary purification of a protein antigen apparently specific to mycobacterium kansasii

A highly anodal protein antigen of molecular weight approximately 10,000 daltons has been identified in culture filtrates of Mycobacterium kansasii and not in culture filtrates of 9 other mycobacterial species. Using immunoabsorbent affinity chromatography, this antigen has been purified and used to skin test guinea pigs sensitized with Mycobacterium kansasii and Mycobacterium tuberculosis. In these animals it was found to be tuberculin active and to have partial species specificity.     

结核分枝杆菌Hsp16.3的表达、纯化和鉴定

目的克隆结核分枝杆菌hspx(acr、Rv2031c)基因,扩增后测序,并在大肠杆菌中表达获得基因重组蛋白Hsp16.3(Heat Shock Protein16.3)。方法用PCR方法从结核分枝杆菌H37Rv基因组扩增出hspx基因片段,连接进入pTA2载体中,序列测定正确后,将其亚克隆到表达载体pProEX HTb并在大肠杆菌DH5α中表达,表达蛋白SDS-PAGE分析后,分别与6×His mAb、16k Da mAb和结核病人血清进行Western-blot,最后用Ni-NTA进行亲和层析纯化蛋白。结果获得结核分枝杆菌Hsp16.3蛋白基因,经序列测定与GenBank公布的序列完全一致;表达蛋白经SDS-PAGE分析,相对分子质量与文献报道一致;Western-blot结果显示,在相对分子量约16kDa处有与6×His mAb和鼠抗16k Da mAb的特异性结合带,而与结核病人血清杂交没有出现结合带。表达产物为包涵体,通过Ni-NTA纯化系统,可得到纯化的目的蛋白。结论成功地表达、纯化和鉴定了Hsp16.3蛋白,它有可能作为新型结核疫苗的靶抗原。     

结核分枝杆菌H37Rv菌株Hsp16.3基因缺失突变株的构建与鉴定

目的 构建及鉴定结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因缺失突变株。方法 体外培养结核分枝杆菌国际标准强毒株H37Rv菌株,并提取基因组DNA,扩增Hsp16.3基因两侧序列,分别插入到pKO质粒载体预定位点中,构建Hsp16.3基因置换型打靶载体,电转入结核分枝杆菌H37Rv菌株内,并与其基因组中的Hsp16.3基因同源交换,筛选出Hsp16.3基因缺失突变株。结果 通过卡那霉素筛选和蔗糖反筛选及PCR鉴定,并在含有潮霉素培养基上不能生长的菌株为Hsp16.3基因缺失突变株。结论 成功构建出结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因缺失突变株。     

Purification and initial characterization of a potential plant vacuolar targeting receptor.

Clathrin-coated vesicles are known to be involved in the transport of proteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of approximately 80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum sativum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the approximately 80 kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the approximately-80 kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.     

Promiscuous peptide of 16 kDa antigen linked to Pam2Cys protects against Mycobacterium tuberculosis by evoking enduring memory T-cell response

One of the main reasons considered for BCG failure in tuberculosis-endemic areas is impediment by environmental mycobacteria in its processing and generation of memory T-cell response. To overcome this problem, we developed a unique lipopeptide (L91) by linking the promiscuous peptide (sequence 91-110) of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys. L91 does not require extensive antigen processing and generates enduring Th1 memory response. This is evidenced by the fact that L91 significantly improved the activation, proliferation, and generation of protective T cells. Furthermore, L91 surmounts the barrier of major histocompatibility complex polymorphism and induces better protection than BCG. This peptide has self-adjuvanting properties and activates dendritic cells. Importantly, L91 activates T cells isolated from purified protein derivative-positive healthy volunteers that responded weakly to free peptide (F91). In essence, L91 can be a potent future vaccine candidate against tuberculosis.     

Lung and blood mononuclear cell responses of tuberculosis patients to mycobacterial proteins.

The differences in specificity of human lung and peripheral lymphocytes for mycobacterial antigens (Ag) need to be evaluated in order to identify vaccine candidates against pulmonary tuberculosis (TB). Therefore, the present study examined the response to low molecular weight secretory proteins of Mycobacterium tuberculosis in bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMCs) from minimal pulmonary TB and non-TB patients. Ag85A, Ag85B, culture filtrate protein (CFP)-31, CFP-22.5, CFP-21, M. tuberculosis protein-64 and an as yet uncharacterised 19 kDa protein were found to be predominantly recognised by BAL cells of TB patients on the basis of lymphocyte proliferation and significant interferon-gamma release. However, recognition of CFP-8, 6-kDa early secreted antigenic target, CFP-10, CFP-14.5, M. tuberculosis secretory protein-17 and five other as yet uncharacterised low molecular weight polypeptides was found to be high on the basis of lymphocyte proliferation at the level of PBMCs. Furthermore, BAL macrophages, and not blood monocytes, were found to produce nitric oxide (NO) in response to mycobacterial Ags. Among polypeptides predominantly recognised by BAL lymphocytes, only Ag85A and Ag85B were found to induce both NO and interleukin-12 (p40) by alveolar macrophages. In conclusion, the present results indicate heterogeneity in antigen recognition by bronchoalveolar lavage cells and peripheral mononuclear blood cells of minimal tuberculosis patients, and also suggest the utility of antigen 85 complex polypeptides for the development of a future mucosal antituberculous vaccine.     

Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis

In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38 kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38 kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38 kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.