Patent Application
20110117134
1. Field of the Invention
The invention relates to a bacterial antigen library, its preparation and use. The library comprises 205 surface protein genes of Brucella suis constructed in an expression vector.
2. Brief Description of Related Art
Brucella, an aerobic, non-spore forming, non-motile Gram-Negative coccobacillus. Brucellosis typically occurs in humans after ingestion of unpasteurized dairy products or after occupational contact with infected humans.
Medical defense against the bio-terror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans.
Until now, there has not been any research group or person that has published or developed recombinant constructs that may be directly used to express all 205 surface proteins of B. suis.
The present inventors have developed a library of a set of putative Brucella surface antigens that can be used by researchers to develop new human vaccines to prevent brucellosis, or diagnostic tests or kits to detect Brucellosis. The proteins can be used to prepare a protein array to investigate pathogenic mechanism of brucellosis.
The complete library of Brucella surface antigens was prepared in a 96 well format and used for an antigen array to screen infectious specimens such as serum and blood cells for the presence of Brucella. The geometric format of the surface antigen array provides a direct technical platform to develop vaccines, methods and drugs to protect people from infectious disease.
The recombinant cells are important and valuable for the following reasons. They allow expression of a bacterial antigen library that is different from any other known library and provide definite genes and proteins used for screening new recombinant vaccines and drugs. This is also a novel technical platform to develop new diagnostic agents and vaccine candidates. This library contains a large set of surface antigens of Brucella for specific diagnosis of infection. The, recombinant proteins provided from this library include purification handles of 6-His and V5 that permit arrangement in an arrayed format or adherence to solid supports that promote their usefulness for other large-scale preparative and screening projects. The array design lends itself to further analysis using bioinformatics approaches. The library will be a valuable resource. These plates can be directly provided to scientists of universities, institutes, and companies for their screening and array studies. Any individual, specific antigen can also be prepared on a large scale from the recombinant library and purified using convenient 6-His and V5 purification handles expressed on each protein.
Therefore, the present invention provides a gene library of putative Brucella suis surface antigens.
The present invention also provides a method of preparation of a gene library of Brucella suis surface antigens.
The present invention permits the cloning and expression of a set of 205 genes of Brucella suis surface protein into expression vectors.
The present invention provides a set of recombinant constructs of Brucella suis surface antigens in 96 well format for further serum and cellular screening.
The present invention provides a method of preparing a vaccine using the gene library of Brucella suis surface antigens.
The present invention provides antigens for diagnosis and prophylaxis against brucellosis.
These features and others will become apparent upon reading the detailed description.
The invention relates to a complete bacterial antigen library of Brucella suis surface antigens, its preparation and use. The library comprises 205 surface protein genes of Brucella suis constructed in an expression vector. The library can be used to prepare recombinant Brucella surface antigens. The expressed proteins derived from the gene library are important for the development of new vaccines to prevent brucellosis, or diagnostic kits to detect brucellosis. The proteins prepared from the gene library can also be used to prepare a protein array to investigate pathogenetic mechanisms of brucellosis.
From the genomic information of the complete sequences of B. suis, 205 open reading frames that encode surface antigens of Brucella were defined and selected. These putative Brucella surface antigens were identified as surface antigens by running prediction algorithms. These antigen genes were PCR synthesized and then constructed into an expression vector, pET101/D-TOPO (Invitrogen Life Technologies, California). The recombinant constructs were transferred into E. coli TOP10 cells (Invitrogen Life Technologies, California) for amplification. The amplified pET-B-SAs encoding surface antigens were collected and purified from the cells. Recombinant plasmids were confirmed to contain surface antigen sequence using PCR method. The positive recombinant E. coli cells are stored at −80° C. in two 96 deep well plates containing LB media with 15% glycerol and 100 ug/ml ampicillin.
We report the results of the use of a Highly Efficient Cloning and Expression System (herein after referred to as HECES) developed by the inventors for attempted expression of a set of 205 B. suis ORFs predicted to encode SPs (surface or secreted proteins). We find that this application of the HECES, which employs methods designed to minimize adjustment of production conditions for individual proteins, results in expression of 71% to 91% of the ORFs, depending on the specificity of the criteria used to demonstrate the presence of protein. Proteins were prepared at high purity in an assay-friendly, microwell, fixed-support format.
The following is a flow chart of the method of HECES.
Preparation of Glycerol Stock of Each Recombinant Cells.
The HECES allows expression of large numbers of bacterial proteins based on genomic sequence information. Purified proteins are produced with high efficiency in a microarray format conducive to analysis of their sero-reactivity against serum from immunized animals. This method is applicable at either small or large scale of protein processing. In this study, we used the Invitrogen directional pET 101/D-TOPO cloning system instead of the Gateway cloning system, but other methods are as previously described.
Prediction of B. suis surface proteins (SPs): A bioinformatic screen of the complete B. suis predicted proteome was undertaken to identify putative surface exposed or secreted B. suis proteins. The bioinformatic pipeline ran a series of searches and analyses including prediction of type I and type II signal peptidase cleavage sites using SignalP, HMM and Blast searches for homology with known outer membrane proteins or protein families, transmembrane segment (TMS) prediction using TMHMM, motif searches for choline, fibrinogen and integrin binding domains, and the identification of repetitive amino acid domains. These data were collated and a set of 205 putative SPs was selected for protein expression. Proteins were included in the set if they had fewer than 3 predicted TMSs and one or more other characteristics supporting their prediction as SPs. Primer design and subsequent gene cloning and protein expression were conducted based on the bioinformatic screen predictions. Subsequently, complete annotation of the genome [5] resulted in re-assignment of the locus tags or alternative start or stop positions for some of the predicted genes. Predicted SP genes are listed in Table 3 by nucleotide position and locus tag ID based on the published annotation. The gene sizes of the Brucella suis surface proteins range from 93 to 3,000 base pairs.
Preparation of B. suis DNA: One milliliter of B. suis strain 1330 (ATCC 23444) bacteria was collected in a 50 ml tube and treated in an equal amount of phenol. After vortexing, the bacterial cells were spun down at 1,560 g, for 10 minutes at 4° C. Nine hundred μL of the supernatant was transferred into a 1.5 mL tube and 630 μL isopropanol was added. The sample was centrifuged at 16,000 g, for 10 minutes at 4° C. The pellet was washed with 70% alcohol and resuspended in 100 μL of sterile water.
Layout of the SP banks and PCR synthesis of B. suis SP genes: Two hundred and five genes that encoded surface protein of B. suis were selected with bioinformatic screen procedure for the gene synthesis. Primer sequences used for the surface antigens were designed and prepared in three 96 well plates as described in Example 1. The entire process for construction of the SP set from predicted gene sequence was implemented in a 96-well plate format to facilitate use of high-throughput methods wherever possible. The predicted SP genes, with their genome position, locus tag, predicted gene size, and their locations in three 96-well plates are shown in Table 3. The forward primers were designed with a CACC sequence at the 5′ end followed by the first 25 bases of the SP gene beginning with the start codon. The reverse primer was comprised of the last 25 bases of the SP gene prior to the stop codon. The PCR reaction was conducted in a final volume of 50 μL that contained 5 μL of 10× ThermalAce reaction buffer, 1 μL of 50 mM dNTP, 14 of 1:10 diluted B. suis template, 0.5 μL of each 50 μM primer, 41 μL of PCR water and 1 unit/μL of ThermalAce. Reactions of B. suis template without primers were used as a negative control. The lacZ gene with primers provided from Invitrogen was used as a positive control. The PCR running protocol was according to our previous study [13] designed as 1 cycle of 95° C. for 2 minutes, 30 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 2.5 minutes, 1 cycle of 72° C. for 10 minutes and then refrigeration at 4° C. The presence and size of PCR products in each well were detected by 1% agarose gel electrophoresis with a 1 Kb DNA ladder (Invitrogen Inc) standard.
Cloning of surface genes into an expression vector: The PCR products were directly cloned into vector pET101/D-TOPO (Invitrogen, Inc). This vector includes a directional cloning site, a T7 lac promoter for high-level IPTG-inducible protein expression, coding sequences for ampicillin resistance, and coding sequences for C-terminal V5 epitope and hexahistidine (6His) expression. Reactions were performed according to the manufacturer's instructions. One ul of PCR product plus 1 ul of water was mixed with 0.5 ul of each salt solution and vector. Ligation reactions were conducted in 96 well plates at room temperature for 5 minutes according to the manufacturer's instructions. One μL of PCR product plus 1 μL of water were mixed with 0.5 μL of each salt solution and vector. The negative and positive transformation controls were pET101/D-TOPO vector alone and pET101/D-TOPO-LacZ, both from Invitrogen. Plasmid transformation was then conducted in 96 well plates. Two μL of the ligation reaction mixture was added to 40 μL of TOP10 competent cells per well and incubated at 4° C. for 30 minutes. After heating at 42° C. for 30 seconds, 250 μL of culture medium was added and the plates were shaken at 37° C. for 1 hour. After this incubation, 20 μL of the contents of each well was streaked on individual wells of 18 pre-warmed 12-well culture plates containing trypticase soy agar with 100 μg/mL ampicillin for clone selection. After plates were incubated at 37° C. overnight, a single colony from each well of the 12-well plates was picked and cultured in 1.2 mL LB broth medium with 100 μg/mL ampicillin in a 96-well deep-well plate for propagation of the recombinant plasmids at 37° C. overnight. On the next day, the plasmids were purified using a 96-well turbo Miniprep Spin column (Qiagen, Inc) and detected by electrophoresis on 1% agarose gel. The presence of inserted genes in the plasmids was detected by PCR using primers designed based on the sequence of the T7 promoter priming site (T7 forward: TAATA CGACT CACTA TAGGG) and the T7 reverse priming site (T7 Reverse: TAGTT ATTGC TCAGC GGTGG).
Expression of recombinant SPs: Competent BL21Star (DE3) E. coli cells were prepared with calcium for expression of B. suis SPs. Four μL of each purified plasmid preparation was mixed with 50 μL of expression host cells in a 96-well deep-well plate and incubated at 4° C. for 30 minutes. After heating at 42° C. for 45 seconds, 250 μL of culture medium was added into each well and the culture shaken at 37° C. for 1 hour. Then, 1 mL of LB solution containing 100 μg/mL of ampicillin was added and the culture was shaken at 37° C. overnight. On the next day, 250 μL of each culture was transferred into 5 mL of LB medium containing 1% glucose and 100 μg/mL ampicillin in 12 well plates and shaken at 37° C. for another 2 hours. Preliminary experiments indicated that this procedure would result in OD600 readings of the cultures between 0.4-0.8 units. IPTG (Invitrogen) was then added to a final 1 mM concentration and the plates were shaken for 5 hours at 37° C. After protein induction, cells were centrifuged at 1,560 g at 4° C. for 10 minutes. The cell pellets were dissolved in 500 μL of cell lysis buffer B-per solution (PIERCE Inc) for detection of protein. Protein expression was examined by both ELISA and Western blot methods.
Plate purification and detection of recombinant SPs by ELISA: A HisGrab plate (Pierce) was used to purify the recombinant surface proteins. Ten μL of the cell lysate and 90 μL of blotting buffer (10 mg casein in 900 mL of PBS, pH 7.4) were placed in wells of triplicate 96-well HisTag antibody plates (EMD Bioscience) and incubated at 4° C. overnight. After each well washed with 250 μL of PBS, 100 μL of a 1:2000 dilution of anti-V5 monoclonal antibody conjugated with alkaline phosphatase (Invitrogen Inc) was added to each well and incubated at 4° C. for 16 hours to detect the recombinant proteins. After the incubation, the plates were washed three times with 1×PBS before 100 μL of developing buffer containing 1 mg/mL Naphthol AS-MX phosphate disodium salt (Sigma) was added to each well. The plates were incubated at room temperature for 1 hour. Light transmission was measured at 410 nm single wavelength and the mean OD of corresponding wells in triplicate plates was taken as a semi-quantitative measure of protein concentration. The positive control was recombinant LacZ tagged with V5/6His and the negative control was medium alone. The OD of wells containing lysates from cells transfected with empty vector was less than 0.05. An OD reading of greater than 0.1 was considered to indicate the presence of recombinant protein.
Protein synthesis with TNT coupled reticulocyte lysate method: Brucella SPs that were not expressed by IPTG induction were synthesized by the TNT coupled reticulocyte lysate method using a reaction kit (Promega). One μg of pET101-SP plasmid was used as the DNA template. Twenty five μL TNT coupled reticulocyte lysate, 1 μL TNT RNA polymerase, 1 μL amino acid mixture (1 mM) and 1 μL RNasin (40 U/μL) were mixed in a 1.5 mL microcentrifuge tube and incubated at 30° C. for 90 minutes. The protein products were detected with WESTERN BLOT.
Column purification of recombinant SPs: Recombinant SPs were affinity purified from cell lysates via their 6His tag under native conditions using the Ni-NTA Superflow 96 BioRobot kit (Qiagen) according to the manufacturer's instructions. The cell pellets were resuspended in 100 μL of B-per solution (Pierce) and kept at room temperature for 30 minutes, then frozen and thawed 3 times. Soluble proteins went through a filter and were added to the Ni-NTA column. Insoluble proteins that were not solubilized in the B-per buffer were treated with inclusion body solubilization buffer (Pierce) and then added to the Ni-NTA column. After the columns were washed twice with NPI-20 buffer, recombinant proteins were eluted with 1 mL NPI-250 buffer into new 96-well deep well plates and stored at 4° C. until further analysis by Western blot.
Preparation of Rabbit Anti-Brucella IgG: Rabbit Anti-Brucella IgG was Used for screening of the recombinant Brucella surface proteins. Rabbits were immunized intramuscularly with dialyzed cell lysate of B. melitensis rough mutant WRR51 [14]. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. Rabbits were immunized intramuscularly with two doses of vaccine (25 μg bacterial lysate per dose) given 4 weeks apart. Control rabbits were injected intramuscularly with the same volume of normal saline. Blood was collected from the rabbits 2 weeks after the second dose of vaccine. IgG was prepared from the serum by affinity chromatography on protein G-Sepharose (Pharmacia-LKB Biotech), adjusted to 1.8 mg/mL, and used to screen SP for antigenicity by Western blot. (Antibodies elicited by immunization with B. melitensis also recognize B. suis proteins.)
Detection of recombinant SP by Western blot analysis Seven μL of each column-purified recombinant SP was mixed with an equal amount of 2×SDS loading buffer. The samples were boiled for 10 minutes and purified on 13% acrylamide/bis gel, then transferred onto nitrocellulose membrane and hybridized overnight with 1:2000 AP-conjugated anti-6His. The membranes were developed with 1 mg/mL of Naphthol AS-MX phosphate (Sigma) and 2 mg/mL Fast Red TR salt (Sigma) in 50 mM Tris buffer.
Screening of SP by Western blot with anti-Brucella antibody. Western blot assay was conducted as described above, except that rabbit anti-Brucella IgG was used.
Synthesis of B. suis SP genes by PCR Probable B. suis SP genes were predicted bioinformatically as described in the Materials and Methods, and a set of 205 predicted SP genes were selected for amplification, cloning and expression. The complete set of these SP is described in supporting information, Table 3.
Molecular sizes of the predicted SP genes ranged from 93 by to 3000 bp. Purified genomic B. suis DNA prepared via phenol/chloroform isolation and detected on 1% agarose gel was used as the template for PCR synthesis of the SP ORFs. In preliminary experiments, we compared four DNA polymerases, pfx, pfu, Taq and ThermalAce (Invitrogen). All the polymerases produce blunt-end PCR products and perform 3′ end proofreading. ThermalAce provided the highest number of positive PCR products and was used to produce the data depicted in
Construction of an expression library of B. suis SP genes: The contents of all 205 wells of PCR-synthesized Brucella SP genes, whether product was demonstrated by agarose gel electrophoresis or not, were directly cloned into an expression vector, pET101/D-TOPO (Invitrogen, Inc), and propagated in TOP10 cells according to the manufacturer's instructions as described in the methods. Recombinant cells were selected on 18 12-well culture plates of LB agar containing 100 μg/mL ampicillin. One well was used for one SP. In the first round of cloning, 201/205 surface antigens (98%) resulted in isolated colonies on the selection plates. Another two rounds of cloning were conducted for the remaining four genes, but not even a single colony was obtained from these clonings. All 198 genes with PCR product detected by agarose gel electrophoresis were present in the set of 201 cloned genes.
A single colony from each well was picked and cultured for plasmid isolation in LB broth. Plasmids from the cultures were purified with a Turbo miniprep method and examined by PCR analysis using forward and reverse primers based on T7. The PCR products were examined by electrophoresis on a 1% agarose gel and 185 plasmids out of 205 SP genes (90%) were found to have inserts with the correct molecular size (
Protein expression and purification: After IPTG-induced recombinant protein expression, the presence of recombinant proteins was assessed in crude cell lysates by both ELISA for detection of the V5 tag and Western blot analysis for detection of the 6-His tag (Table 3). Recombinant LacZ protein fused with 6His N5 tags was used as a positive control for both assay methods. An OD value of 0.10 in the ELISA was used as the minimum value that indicated the presence of recombinant protein, indicated by a P in Table 3. Proteins that showed a visually detectable band on Western blot are marked with a P in the respective column and those that showed no band are marked N; a question mark indicates that 10 proteins showed bands of a size different than that predicted from the ORF analysis. The number of expressed recombinant SP detected by these methods is summarized in Table 1.
aSP present in inclusion bodies
Proteins were detected by ELISA in 171/205 wells (83%) and by Western blot in 181/205 wells (88%). Proteins were detected in 167/205 wells (81%) by both methods and in 185/205 wells (90%) by either method. Fifteen SPs were positive only by Western blot and 3 were positive only by ELISA. A total of 20 out of 205 wells (10%) were negative by both ELISA and Western blot. The percentage of positive wells detected by one, both or either method was generally similar across all three plates. Of the 181 recombinant proteins detected by Western blot, 31 represented proteins present in inclusion bodies.
The 20 plasmids that did not express SPs were used as templates to conduct TNT protein synthesis. Three expressed genes (marked C in the TNT column of Table 3) were used as positive controls to monitor the TNT protein synthesis; all 3 produced recombinant proteins. Of the 20 non-expressed genes, 7 (marked with a P in the TNT column) produced proteins by the TNT method. After the presence of protein was confirmed, cultures from positive wells were expanded and stored in 20% glycerol in 96 well deep well plates and referred to as the B. suis SP protein bank.
Screening recombinant proteins for immune reactivity against anti-Brucella rabbit IgG: We investigated the Brucella-specific seroreactivity of the 167 recombinant Brucella proteins detected by both anti-6His Western Blot and anti-V5 ELISA, and two additional proteins selected. The 169 selected proteins were column purified and concentrated after expression from the B. suis SP protein bank as described in the methods. Five of the expressed proteins (Table 2) were detected by Western blot analysis using rabbit anti-Brucella IgG. SodC is a periplasmic protein in B. abortus [15]. None of the other proteins have been demonstrated to be cell surface or secreted proteins.
Brucella-Reactive Recombinant SPs
It is obviously difficult, even impossible, by using traditional cloning technologies to construct the enormous number of genes from the simplest organism and evaluate their multiple functional products within a short time. The High Throughput (HT) concept has provided useful technologies that will be necessary to harness the informatic power of genome sequencing. We have previously described the development of conditions for the HECES HT method [13]. In this report, we have modified and extended the HECES to express and screen the Brucella SPs as a model approach to whole-genome functional screening. We expressed a high proportion of the selected genes using pre-defined conditions that were applied across the entire gene set. The HECES includes three iterations of PCR cloning at pre-set conditions, with descending stringency based on annealing temperature. This approach led to full-length cloning of 198/205 genes (96.6%) with no requirement to adjust individual conditions for each gene.
As expected, HT protein expression was more difficult, given the variety of challenges encountered in translation, solubilization, secretion and potential protein toxicity for the expression host. In addition to these issues, which may affect the production of proteins of correct sequence, proper protein folding is required for a number of functional tests. By their nature, HT approaches do not permit much flexibility in tailoring reaction conditions to optimize expression of each protein in a conformationally correct form. We focused on protein solubility as an important determinant for extraction from the host cells and separated proteins first with non-denaturing conditions and then used denaturing conditions for insoluble proteins. Extraction using denaturing conditions resulted in purification of 31 of the 181 proteins that were positive by Western blot and increased the overall protein yield by 15% (31/205 genes). The modified HECES we used in this study was highly successful for construction of the SP205 protein library. Overall, protein was expressed from 81% to 90% of in silico-predicted SP genes (depending on the criteria used for positivity) using only two variations in expression and extraction conditions. The higher number may be more accurate, since the quantity of protein in some of the ELISA-positive wells may have been too little to detect by WESTERN BLOT. The failure of protein expression did not appear to be related to gene size (data not shown).
HT expression of protein from a higher percentage of genes should be possible, but cost may increase considerably. Interestingly, when we attempted in vitro TNT translation from the genes that did not express proteins in BL21 Star cells, only 7 (35%) yielded products, while all three of the control genes were expressed. Although this study was not primarily designed to assess the relative merits of in vivo and in vitro translation, this observation suggests that use of in vivo translation at the single set of conditions we chose would not overcome all the limitations of our in vivo approach. Although the addition of in vitro translation as a conditionally programmed HT step (instead of ad hoc as we did) may be useful to increase the number of expressed proteins, the added benefit in our study would have been only 3.4% (7/205 genes). The value of additional iterations of in vitro translation with variations in experimental conditions or further optimization of the conditions could be evaluated in later studies. The HECES would provide an excellent benchmark for assessing the relative efficiencies of in vivo and in vitro translation systems under a variety of experimental conditions for a large protein set.
This project was originally designed to produce proteins that were arrayed in a format suitable for discovery of vaccine or diagnostic antigens suitable for further development to combat brucellosis. For analysis of Brucella seroreactivity, we initially planned to capture proteins from the crude lysate directly on an ELISA plate by use of either chelation of 6His to nickel or by antibody-mediated binding of the V5 tag. In preliminary studies (data not shown), we found that there was variability in binding using the two different methods. After applying the same amount of purified recombinant proteins on the two kinds of plates, OD readings over baseline on the anti-6His antibody plate were at least 2 fold higher than those observed with the Nickel-chelating plate. Due to this variability, we included a Ni-NTA column chromotography step to purify and concentrate proteins prior to probing with anti-Brucella IgG. We used native conditions for the purification, since our primary intent was to screen the library for immunogenicity with antibodies, which optimally requires maintenance of the native protein conformation. Using this column, we obtained approximately 20 to 300 μg per 5 mL bacterial culture. This large range of protein yield may reflect the wide range of protein solubility and binding properties as well as variability in protein expression under relatively uniform conditions. For production of libraries for purposes that do not require retention of native protein conformation, denaturation and purification via the 6His tag should be considered as it may yield a higher success rate. For our purpose, the absolute quantity of protein was not crucial, since only small amounts were required for studies of antigenicity.
In preliminary studies (data not shown), we arrayed the proteins on two microtiter plates after adjustment for protein concentration. Plates were screened by ELISA using the rabbit antibody employed for the anti-Brucella Western blot and compared with IgG prepared from non-immune rabbits. Although a number of additional proteins were reactive in this assay, there was significant variability among repeat experiments. Additional experiments are in progress to refine this assay for plate-based HT screening. Screening of all 167 SP by Western blot resulted in detection of five proteins, one of which (SodC) has been localized in the periplasmic space of B. abortus [15]. These Western blot results confirmed the utility of the HECES for HT cloning and expression of immunoreactive Brucella proteins. The five proteins we identified, as well as the likely additional immunoreactive proteins, may have utility as diagnostic or vaccine antigens for brucellosis.
The technology platform used to produce the large number of Brucella proteins in this study is a modification of the HECES we previously reported [13]. The HECES was designed to be compatible with either automated systems or benchtop methods using a 96-well plate format. In our previous report, we used Invitrogen's TOPO DNA topoisomerase I system for cloning PCR products into a Gateway entry vector, followed by cloning into a Gateway destination vector. For preparation of the SP205 protein library, we improved the HECES by cloning PCR products directly into an expression vector using the TOPO directional cloning system. This approach reduced the number of steps in the overall process, with associated reduction in time and cost. This single-step strategy not only saves working time but also avoids many potential problems that may impact the cloning success rate. The Gateway system is highly flexible for later application of the gene library, since it allows use of different destination vectors and promoters that can be applied to different hosts. This limitation is counterbalanced for our requirements by the improved efficiency in reaching the main goal of developing the protein library.
Our success rate in expression of the SP205 protein library is consistent with our previously reported success rate of 90% (18/20 proteins) using the Gateway-based HECES [13] for preparation of proteins of varied size and functions. The tradeoffs between high throughput and the need to individualize reaction conditions are most evident at the protein expression and purification steps. The SP205 protein library represents a reasonable compromise among cost, time and utility. Although it is likely that additional seroreactive proteins may be present in the remainder of the complete B. suis ORFeome), the positive results we obtained are sufficient to enable a number of additional studies directed toward development of new diagnostic tests and vaccines using the putative surface antigens identified and enumerated herein.
The recombinant constructs can be used to express a complete set of putative Brucella surface antigens. The recombinant constructs are amplified and purified from their recombinant cells. Both recombinant constructs and cells are prepared in a 96 well plate format by direct transferring between multi-well plates. These plates can be used as final products directly to provide to scientists of universities, institutes, and industrial companies for their specific screening and array studies. Once any individual, specific antigen is selected during the studies, these antigens can also be prepared in a large scale from the recombinant library and purified using convenient 6-His and V5 purification handles located on C terminal of each recombinant constructs. The plates that contain either recombinant constructs or cells can be fast prepared with low coast and be easily storage.
Table 3 shows detection and anti-Brucella seroreactivity of recombinant B. suis SPs.
aLocus Tag entries based on published annotation; missing entries indicate that locus tags were not applicable because of alteration to the genome annotation subsequent to the original locus assignment.
dWB against 6His positive = P, negative = N, showed bands of a size different than that predicted from the ORF analysis = ?.
cPresent in inclusion bodies = Y.
dExpressed in TNT expression system = Y.
eDetected by WB using IgG from rabbits immunized with rough B. melitensis WRR51 = Y.
Determination of B. Suis Surface proteins, 205 genes of B. suis SP were selected and their size shown in 96 well format as follows:
Purification of plasmid: Transfer 100 ul bacteria/well to a new deep plate and add 500 ul of LB culture solution with 100 ug/ml Ampicillin. Culture at 37° C. overnight for preparation of glycerol stock. The plasmids were purified using 96 turbo miniprep method (Qiagen, Inc). The 0/N cell cultures were centrifuged at 3,000 RPM for 10 min at 4° C. The supernatants were decanted and cells kept for following processing.
Transfer supernatants to Turbo filter and vacuum to plasmid filter. Wash the filter with 700 ul PB/well and vacuum. Wash the filter with 700 ul PE/well and vacuum. Centrifuge the filters at 3,000 RPM for 10 min at 4° C. Add 200 ul EB to each well and centrifuge the EB to a 96 well plate at 3,000 RPM for 10 min at 4° C.
A method of identifying and expression Brucells suis surface antigens proteins was conducted. A bioinformatics screen of the complete Brucella suis genome was made to identify genes that encode putative surface or secreted proteins. This was done by designing primers to enable PCR amplification of predicted genes with appended sequences; arraying the primers and conducting cloning reactions in a multiwell microplate format suitable for liquid handling with multichannel devices; cloning of the predicted genes into E. coli.; purifying of plasmids containing the desired sequence; verifying of correct insert size and sequence in purified plasmids; storing of plasmids and E. coli containing vector with cloned genes; (Gene bank) cloning of genes into expression vector and storage of expression plasmids and E. coli containing expression vector with cloned genes; (Protein bank); inducing of protein expression from protein bank; and verifying the expressed protein size. The results of this are presented herein. The primers used are presented herein.
A method of detecting antigenic proteins of Brucella suis was conducted by creating a library of Brucella suis surface antigens. We found 205 surface antigens. The antigenicity of Brucella suis proteins was diagnosed by comparing a sample from a patient or immunized or infected experimental animal to the library to determine if Brucella suis antigens were recognized. When Brucella suis was recognized, we were able to diagnose antigenicity of the proteins.
Diagnosing exposure to Brucella suis can be conducted by creating a library of Brucella suis surface antigens; diagnosing exposure to Brucella suis by comparing reacting a sample from a patient to with said library to determine if Brucella suis antigens are presentrecognized; and diagnosing exposure if said antigens are recognized.
Primers: The following primer sequences were used:
SEQUENCE LISTING: The complete nucleotide sequences of the putative surface and secreted protein genes amplified by PCR from B. suis DNA are listed below.
The invention described herein may be manufactured, used and licensed by or for the U.S. Government.
