Identification of seroreactive proteins in the culture filtrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn's disease patients

The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA ( P <0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.     

Identification of proteins of potential diagnostic value for bovine paratuberculosis

Previously we showed that Mycobacterium paratuberculosis culture filtrates (CFs) contain more antigens that react with sera from infected cattle than do cellular extracts of the organism. The goal of the present study was to identify proteins of potential diagnostic value among these CF proteins. Proteins of potential interest were first separated by 2-DE. Roughly 240 CF protein spots were detected on CBB-stained gels using Phoretix 2D software. Of these, 83% reacted with serum from M. paratuberculosis-infected cattle in immunoblots. When bovine serum was absorbed with M. phlei antigens, however, only 37 of these antigenic protein spots were reactive. Twenty-four of these spots were selected for identification based on their immunoblot staining intensity and differences in pI and mass. A total of 14 proteins were ultimately identified by MS and BLAST searches as ModD, PepA, ArgJ, CobT, Antigen 85C, and nine hypothetical proteins. N-terminal peptide analysis of PepA, Antigen 85C, ModD, MAP1693c, MAP2168c, and MAP1022c showed that each protein has 27-39 amino acids that may function as a signal sequence suggesting they are secreted through a Sec-dependent pathway. These 14 proteins from M. paratuberculosis CF are strong candidates for use as antigens for improved serodiagnostic tests for bovine paratuberculosis.     

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10⁴-10^− 1) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10² and 10³ MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1 MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6 weeks by real-time PCR.     

Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli

Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.     

结核分枝杆菌Rv3871抗原优势肽段的克隆表达及抗原性鉴定

目的:为了提高结核病诊断试剂的特异性和敏感性,克隆表达结核分枝杆菌H37Rv株RD1区Rv3871抗原优势肽段,并应用ELISA法对其抗原性进行初步鉴定。方法:利用Biosun生物信息学软件对Rv3871抗原进行表位分析,通过PCR从结核分枝杆菌H37Rv基因组中扩增Rv3871抗原优势肽段编码基因,在大肠杆菌HB101中进行表达,采用间接ELISA法初步评价其抗原性。结果:Rv3871-1肽段检测的敏感性为32.5%(13/40),特异性为97.2%(35/36);Rv3871-2敏感性为45%(18/40),特异性为100%;Rv3871-3敏感性为37.5%(15/40),特异性为91.6%。结论:结核分枝杆菌RD1区Rv3871抗原优势肽段Rv3871-2有较高的特异性和敏感性,有望作为候选抗原用于结核病患者的血清学检测。     

Sero-prevalence of paratuberculosis in young kids using ‘Bison type’, Mycobacterium avium subsp. paratuberculosis antigen in plate ELISA

Two Mycobacterium avium subsp. paratuberculosis (MAP) antigens (native—S 5, ‘Bison type’ and commercial antigens ‘Bovine’), were compared for screening of kids against paratuberculosis infection. Using MAP (S 5) antigen (‘Bison type’) in plate ELISA, 47 serum samples driven from farmer's herds of Jakhrana, Sirohi, and Marwari breeds in their home tract in Rajasthan state were screened. Of the 47 kids randomly sampled, 8.5% were found sero-positive by plate ELISA test. Breed-wise sero-prevalence was 10.5%, 7.6%, and nil in the Jakhrana, Sirohi, and Marwari male kids, respectively. Whereas, none of the serum sample was found positive using commercial MAP ‘Bovine’ antigen. Sero-prevalence of paratuberculosis has been found to be low in young kids (2 months old) belonging to the farmer's herds of Jakhrana and Marwari in their home tracts.     

Evaluation of an indigenous ELISA for diagnosis of Johne’s disease and its comparison with commercial kits

Country lacks sensitive and indigenous diagnostic kits for the screening of goats and sheep against Johne’s disease. Therefore an indigenous ELISA kit was developed using protoplasmic antigen from native Mycobacterium avium subspecies paratuberculosis ‘Bison Type’ strain of goat origin (Kit 1). In the present study, kit 1 and two commercial kits (kit 2 and 3) were evaluated with respect to ‘Gold Standard’ fecal culture in 71 animals (55 goats and 16 sheep). Kit 1 using indigenous antigen (protoplasmic antigen) was sensitive at very low concentration (0.1 μgm / well) as compared to purified commercial protoplasmic antigen (4 μgm / well) used in kit 2, in the Type 1 reactors (strong positive as positive). Screening of 71 animals by fecal culture detected 38.0% animals (goats-40.0%, sheep-31.2%) as positive (clinical shedders of bacilli) from these farm animals. Of the farm animals located at Central Institute for Research on Goats, herds of goat were endemic whereas, sheep flocks were comparatively resistant to Johne’s disease. The 29.5 and 61.9, 15.4 and 57.7 and 4.2 and 14.0% animals (goats and sheep) were in the category of sero-reactors type 1 and 2 of the ELISA kits 1, 2 and 3, respectively. In the type 1 sero-reactors, sensitivity and specificity of kit 1, 2 and 3 was 53.7 and 86.0, 17.8 and 86.0 and 3.5 and 94.7%, respectively. Indigenous ELISA test (kit 1) was significantly superior for the screening of goatherds and sheep flocks against JD as compared to commercial ELISA kits (Kit 2 and 3). In comparison to kit 2 and 3, kit 1 had highest sensitivity, comparable specificity and substantial to nearly perfect proportional agreement (Kappa Scores) with respect to ‘Gold standard’ fecal culture in goats and sheep. Disease being endemic in herds and flocks screened using ELISA kits, Type I sero-rectors had better correlation with fecal culture in comparison to Type II sero-reactors therefore, used for estimation of sero-prevalence. Newly developed Indigenous ELISA kit was simple, inexpensive, sensitive and reliable for screening of goats and sheep population against Johne’s disease. The study reports high prevalence of Johne’s disease in farm goatherds and sheep flocks, using sensitive tests (fecal culture and ELISA kit). Results of Type 1 reaction in kit 1 were optimally correlated with culture and were good for estimating the sero-prevalence. For controlling Johne’s disease in endemic herds initial removal of the animals in strong positive category (Tyep 1 reactors), may help to remove heavy shedders.     

Trypanosoma congolense: expression of a heat shock protein 70 and initial evaluation as a diagnostic antigen for bovine trypanosomosis

A 69-kDa immunodominant protein of Trypanosoma congolense was identified as a member of the hsp70 family that is homologous to mammalian BiP. We report here the expression of the gene encoding the T. congolense BiP in a bacterial system. Dot blotting of the truncated recombinant proteins confirmed that BiP antigenicity is mainly located in the C-terminal third of the molecule. A recombinant fragment corresponding to this region was used as an antigen in an indirect ELISA and an initial evaluation of its diagnostic potential for bovine trypanosomosis was performed. The test showed limited sensitivity for detection of primary-infected cattle but was capable of accurately detecting secondary infections. As BiP and its derivatives may be produced at low cost under stable forms allowing standardization of the tests, they warrant further evaluation as antigens fro serodiagnosis of bovine trypanosomosis.     

Recognition of Human Anti-DNA Autoantibodies by Secretory Antigen 85 Complex of Mycobacterium Tuberculosis H37Rv

Secreted mycobacterial protein antigens were isolated from the culture filtrate of Mycobacterium tuberculosis H₃₇R_v strain grown in Sauton's medium. These secretory proteins of Mycobacterium tuberculosis culture filtrate (MTCF) were separated by SDS-PAGE. High titre anti-DNA autoantibodies from the sera of systemic lupus erythematosus (SLE) patients showed remarkable binding and specificity towards MTCF proteins in dot blot and solid phase immunoassays. The major immunodominant secretory protein in MTCF, fractionated by Sephadex G-200 chromatography was the antigen 85 complex comprising of 30 and 31 kDa molecular weight components. Binding of SLE anti-DNA autoantibodies to the antigen 85 complex was further confirmed by Western blotting. The results suggest the possible involvement of secretory mycobacterial protein antigens in anti-DNA antibody induction in SLE.     

结核分枝杆菌4种抗原的基因克隆、表达、纯化和抗原性初步检测

目的:克隆38kD、ESAT-6、CFP10和MPT64等4种结核分枝杆菌抗原基因,利用大肠杆菌表达系统分别表达重组蛋白,纯化并初步评价其抗原性。方法:通过PCR方法从结核分枝杆菌H37Rv株基因组中扩增38kD、ESAT-6、CFP10和MPT64抗原的基因,连接入pBVIL1表达载体,在大肠杆菌HB101株中进行表达,以间接ELISA方法初步评价其抗原性。结果:获得了结核分枝杆菌抗原38kD、ESAT-6、CFP10和MPT64的基因,并在大肠杆菌中进行了高效表达,初步验证所纯化获得的抗原具有良好的抗原性。结论:pBVIL1表达载体可以高效表达多种结核分枝杆菌抗原,38kD、ESAT-6和CFP10抗原均可作为结核病血清学诊断的候选抗原。     

Development of PLEX, a plasmid-based expression system for production of heterologous gene products by the gram-positive bacteria Streptococcus gordonii

While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.     

Antigen discovery: a postgenomic approach to paratuberculosis diagnosis

Paratuberculosis is a chronic enteritis caused in domestic and wild ruminant species by Mycobacterium avium subsp. paratuberculosis (MAP) that is responsible for major economic losses to the agricultural industry. To date, no satisfactory therapeutic, vaccine, or diagnostic tools are available, globally impairing all control programs. In this study, we have undertaken a large-scale postgenomic analysis of MAP proteins, to identify specific antigens that could potentially improve the diagnosis of paratuberculosis. Two complementary approaches were implemented, the first one consisting in the systematic proteomic identification of proteins present in MAP culture filtrates (CFs), followed by the selection of MAP-specific proteins by BLAST query on available mycobacterial genomes. The resulting database represents the first established secretome of MAP and a useful source of potentially specific antigens. The second approach consisted in the immunoproteomic analysis of both MAP extracts and CFs, using sera from MAP-infected and uninfected cattle. Combining results obtained with both approaches resulted in the identification of 25 candidate diagnostic antigens. Five of these were tested in an ELISA assay for their diagnostic potential, on a limited panel of field sera, and the combination of three of them competed in performance with available commercial assays, reaching a test sensitivity of 94.74% and specificity of 97.92%.     

Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C. difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E. coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E. coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40 mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C. difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C. difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.     

牛呼吸道合胞体病毒G蛋白的截短表达与鉴定

经生物学软件DNA Star分析,将牛呼吸道合胞体病毒G基因截短成2个片段G1和G2。然后用人工合成的牛呼吸道合胞体病毒G基因为模板,用PCR分别扩增G1和G2基因片段,其大小分别为570 bp和308 bp。将目的片段定向克隆到pET30a表达载体中,酶切及测序鉴定均正确后,转化BL21表达菌,经IPTG诱导后G1和G2基因片段都获得了表达,且都为可溶性表达。用Ni柱亲和层析法在非变性条件下纯化重组蛋白,经免疫印迹试验鉴定证明纯化的重组蛋白G1具有良好的抗原性和特异性,而重组蛋白G2无反应性。应用纯化的重组蛋白G1进行的间接ELISA与免疫印迹试验在国内牛血清中检测到了BRSV血清抗体。本研究所表达的重组蛋白G1为基于牛呼吸道合胞体病毒G蛋白的血清学诊断方法的建立与牛呼吸道合胞体病毒G蛋白生物学功能的研究奠定了基础。     

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc-(His)₆ tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested post-translational modifications may contribute to the size difference. The NH₂-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.     

Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.     

Suitability of yeast- and Escherichia coli-expressed hepatitis B virus core antigen derivatives for detection of anti-HBc antibodies in human sera

Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients' sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.     

Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)–amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH₂ (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.     

Development and use of a partial Mycobacterium avium subspecies paratuberculosis protein array

As an initial step toward systematically characterizing all antigenic proteins produced by a significant veterinary pathogen, 43 recombinant Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) expression clones were constructed, cataloged, and stored. NC filters were spotted with purified proteins from each clone along with a whole cell lysate of M. paratuberculosis. Spots on the resulting dot array consisted of hypothetical proteins (13), metabolic proteins (3), cell envelope proteins (7), known antigens (4), and unique proteins with no similarity in public sequence databases (16). Dot blot arrays were used to profile antibody responses in a rabbit and mouse exposed to M. paratuberculosis as well as in cattle showing clinical signs of Johne's disease. The M. paratuberculosis heat shock protein DnaK, encoded by ORF MAP3840 and a membrane protein (MAP2121c), were identified as the most strongly immunoreactive in both the mouse and rabbit hosts, respectively. MAP3155c, which encodes a hypothetical protein, was most strongly immunoreactive in sera from Johne's disease cattle. This study has enabled direct comparisons of antibody reactivity for an entire panel of over 40 proteins and has laid the foundation for future high throughput production and arraying of M. paratuberculosis surface proteins for immune profiling experiments in cattle.