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1 Department of Proteomics, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden
2 Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, SE-752 36 Uppsala, Sweden
3 Department of Bacteriology, National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden
4 The Mycoplasma Group, Veterinary Laboratories Agency (VLA), Addlestone, Surrey KT15 3NB, UK
Correspondence
Anja Persson
anja{at}biotech.kth.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table listing the primers used in PCR, mutagenesis and DNA sequencing is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The use of antibiotics, such as tylosin and tetracyclines, for treatment of CBPP is a much-debated subject. It has been considered ineffective and a potential promoter of silent carriers of the disease (Provost, 1996
). However, newer antibiotics, such as danafloxacin, have been shown to reduce the spread of CBPP (Hübschle et al., 2006). The slaughter of infected herds still appears to be the most effective means to eradicate CBPP (Windsor & Wood, 1998). This strategy has been used in Europe, but the disease has re-emerged in every decade of the 20th century (Nicholas et al., 2000). CBPP is endemic in much of sub-Saharan Africa, and eradication by mass slaughter would be too expensive and have severe consequences (Kusiluka & Sudi, 2003; Windsor, 2000). In Botswana, CBPP was successfully eradicated in 1995 by a stamping-out strategy (Windsor & Wood, 1998) which turned out to be directly correlated with increased malnutrition in children (Boonstra et al., 2001). Extensive vaccination campaigns seem to be the best option for Africa (March, 2004; Windsor, 2000). However, recent vaccination campaigns have given poor results due to weak vaccine effects (Nicholas et al., 2000; Thiaucourt et al., 1998, 2000). The vaccines currently in use are live strains with reduced pathogenicity, and the vaccine currently recommended by the OIE, strain T1-44, has disadvantages such as lack of long-term immunity (Kusiluka & Sudi, 2003), late protection (1–3 months for primo-vaccination) (Thiaucourt et al., 1998) and poor protection in vaccine trials (Thiaucourt et al., 2000). Pathogenicity of T1-44 has also been shown (Mbulu et al., 2004).
Little is known of the pathogenicity of M. mycoides SC. No secreted toxins or surface receptors mediating adhesion or cellular responses in host tissues have been reported. There are factors associated with pathogenesis, although their precise functions are unclear. The galactan capsule, an oligosaccharide layer surrounding the cell membrane, appears to promote binding to host tissues, convey resistance to phagocytosis and have toxic effects, and may cause autoimmune responses by structural similarities to bovine pneumogalactan (Buttery et al., 1976; Nicholas & Bashiruddin, 1995). Oxidative damage to the host tissue from hydrogen peroxide (H2O2) produced by glycerol metabolism in M. mycoides SC (Miles et al., 1991) may contribute to CBPP lesions and has been described for other mycoplasma species (Tryon & Baseman, 1992). Five immunogenic lipoproteins of M. mycoides SC, LppA (Cheng et al., 1996; Monnerat et al., 1999), LppB (Vilei et al., 2000), LppC (Pilo et al., 2003), LppQ (Abdo et al., 2000) and Vmm (Persson et al., 2002), have recently been described; however, their biological functions or possible roles in pathogenicity are, to our knowledge, still unknown.
Many mycoplasmas express surface proteins that undergo reversible changes to alter the antigenic repertoire at the cell and population levels (Citti & Rosengarten, 1997; Razin et al., 1998), as an adaptation to evade the host immune response. Some variable surface proteins are involved in adhesion and immunomodulation (Le Grand et al., 1996; Sachse et al., 2000; Washburn et al., 1993). Only two variable surface proteins are known in M. mycoides SC (Gaurivaud et al., 2004; Persson et al., 2002). One of them, Vmm, is a small lipoprotein of 17 kDa whose expression is regulated at the transcriptional level by the number of (TA)n repeats in the promoter spacer. Fourteen putative proteins with similar promoters are present in the M. mycoides SC genome (Westberg et al., 2004), and are referred to as Vmm-type proteins in this study.
The primary aim of this study was to investigate six of the Vmm-type proteins, to detect whether humoral immune responses are raised against them in vivo, if they are expressed in vitro and if the expression in vitro is variable. An additional aim was the development of an optimized scheme for high-throughput production of recombinant M. mycoides SC proteins in Escherichia coli. This would enable the screening of a large number of proteins for possible selection as target antigens for the development of diagnostic tests and recombinant vaccines.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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)] were selected for this investigation. This subset included Vmm. Their peptide sequences were retrieved from the genome sequence at EMBL/GenBank/DDBJ entry BX293980 and analysed with TMHMM (Krogh et al., 2001) and SignalP (Nielsen et al., 1997; Nielsen & Krogh, 1998) to identify transmembrane regions and signal peptide sequences, respectively, LipoP (Juncker et al., 2003) to predict lipoproteins, and InterProScan (Quevillon et al., 2005; Zdobnov & Apweiler, 2001) to identify protein motifs.
Primer design and expression vector.
PCR primers (see Supplementary Table S1 available with the online version of this paper) were designed to amplify the whole genes except the signal peptide sequences. NotI or AscI restriction sites were added to the 5' end of the primers to allow directed insertion into the vector pAff8c (Larsson et al., 2000). The reverse primer was biotinylated to enable solid-phase cloning, and a 3C protease cleavage site (not used in this study) was included in the forward primer handle to enable removal of the His6–albumin binding protein (ABP) fusion tag of the recombinant protein. The His6 moiety of the fusion tag allows purification by immobilized metal affinity chromatography (IMAC), while the ABP (Nygren et al., 1988) enhances solubility and is immunopotentiating (Libon et al., 1999; Sjölander et al., 1997). Names of the recombinant proteins were derived from the corresponding ORF names (Westberg et al., 2004) from EMBL/GenBank/DDBJ accession number BX293980.
M. mycoides SC strains.
The M. mycoides SC type strain PG1T was grown in F medium (Bölske, 1988). Genomic DNA was prepared and purified by proteinase K lysis and phenol : chloroform extraction. Total RNA was isolated with Trizol reagent (Life Technologies) from 200 ml culture. For colony immunoblotting, M. mycoides SC strains PG1T and M223/90, a pathogenic strain from Tanzania (Bölske et al., 1995), were grown on F medium agar plates.
Cloning.
PCR was performed with 20 ng template DNA using AmpliTaq DNA polymerase (Roche). The biotinylated PCR products were immobilized on Dynabeads M280-streptavidin paramagnetic beads (Dynal Biotech), and the bound PCR fragments were washed and cleaved with NotI (New England Biolabs). The buffer was replaced, and amplicons were released from the beads by AscI (New England Biolabs) digestion. Cleaved fragments were thereafter ligated into AscI/NotI-digested pAff8c plasmid using T4 DNA ligase (Fermentas) and the constructs were heat-shock transformed into E. coli strain BL21(DE3) cells (Novagen). Correct clone sequences were verified by sequencing. Single-stranded DNA template was generated using TempliPhi (GE Healthcare) prior to cycle sequencing with BigDye Terminator chemistry (Applied Biosystems). The sequencing reactions were analysed on an ABI PRISM 3700 sequencer (Applied Biosystems) and the data analysed using Sequencher software (Gene Codes). To substitute the mycoplasma TGA tryptophan codon with the E. coli TGG tryptophan codon, the QuikChange Multi Site-Directed Mutagenesis kit (Stratagene) was used. Plasmid preparations were made with the QIAprep Spin Miniprep kit (Qiagen).
RT-PCR.
For expression analysis, RT-PCR was performed with the SuperScriptIII One-Step RT-PCR system with a Platinum Taq kit (Invitrogen) in 40 cycles. First-strand synthesis was performed with 1 µg RNA and 15 pmol reverse primer at 50 °C for 30 min.
Protein expression and purification.
The recombinant proteins were expressed in E. coli BL21(DE3) and were purified by IMAC (Porath et al., 1975), as previously described (Steen et al., 2006). Purified samples were diluted from 6 to 1 M urea with PBS (2 mM NaH2PO4, 8 mM Na2HPO4, 150 mM NaCl) and were subsequently concentrated on a Vivapore 10/20 concentrator (Vivascience) to a final volume of 1.5 ml. The protein concentrations were estimated with the bicinchoninic acid (BCA) assay, and the protein-50 assay (Agilent Technologies) was used to measure protein purity.
Antibodies and sera used in immunoblotting.
Sera from 15 CBPP cases were used in this study. Four were from an outbreak in Botswana in the middle of the 1990s (G1–4), eight were from an outbreak in Namibia in May 2004 (1MUK15A–17MUK15A), one was an experimental infection from Namibia in 2001 (Exp. Inf.), one serum was from an outbreak in Tanzania in 1997 (PW227), and the final serum was from Kenya in 1998 (C102). For information on CBPP diagnostic test results, see Table 1. Fetal bovine serum (FBS; Gibco) and sera from five healthy Swedish cattle were used as negative controls. The sera were diluted 1 : 400 and 1 : 5000; both concentrations were used for each serum. They were blocked with an E. coli lysate and purified His6–ABP to prevent false signals due to interaction with residual E. coli proteins in the recombinant protein samples or to the His6–ABP tag which originates from streptococcal protein G. For the pre-adsorption experiments, 500 µg of the six recombinant Vmm-type proteins was added to separate 1 ml aliquots of serum 15MUK15A, prepared as described above.
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Immunizations and affinity purification of antibodies.
Rabbits were immunized with the purified recombinant Vmm-type proteins in accordance with national guidelines (Swedish permit A84-02). The protocols for immunization and subsequent affinity purification of antibodies have been described previously (Nilsson et al., 2005). Briefly, the serum was depleted of antibodies reactive to the His6–ABP fusion tag, followed by enrichment of Vmm-type-protein-specific antibodies on an affinity column containing the recombinant proteins, and removal of all other antibodies. Collected antibody fractions were diluted 1 : 1 with 87 % (v/v) glycerol and 0.02 % (v/v) NaN3, and stored at –20 °C. Antibodies were named after the corresponding recombinant Vmm-type protein, e.g. polyclonal antibody (pAb) A117 was produced from recombinant protein R117, etc.
Colony immunostaining.
Freshly grown mycoplasma colonies were transferred to nitrocellulose membranes and blocked in TBST [TBS, 0.05 % (v/v) Tween 20] containing 10 % (v/v) horse serum, followed by incubation with the affinity-purified pAbs (diluted 1 : 50, to 1.5–6.6 µg ml–1) for 1.5 h at room temperature. The Vmm-specific mAb 5G1 (9 µg ml–1) (Brocchi et al., 1993; Persson et al., 2002) was used as a positive control. Bound antibodies were detected with HRP-conjugated swine anti-mouse or goat anti-rabbit IgG (DakoCytomation) at a final concentration of 0.3 µg ml–1 using 4-chloro-1-naphthol as substrate. The membranes were also stained with Ponceau S in order to identify negative colonies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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for a summary of in silico analysis.
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Humoral immune responses to the Vmm-type proteins in CBPP-diseased bovines
In order to analyse whether native Vmm-type proteins are expressed in mycoplasmas during infection, the corresponding recombinant Vmm-type proteins were subjected to dot and Western blotting against 15 bovine sera from four CBPP outbreaks, as well as five sera from healthy Swedish cattle and FBS as negative controls. Dot blotting was performed as an initial screening to allow analysis under non-denaturing conditions. An M. mycoides SC strain PG1T lysate was used as a positive control, and the recombinant fusion partner His6–ABP and an E. coli whole-cell lysate were used as negative controls. The results (Fig. 1, Table 3) showed that all CBPP-positive sera contained antibodies that bound several recombinant proteins. Interestingly, the signal intensities varied between the sera. Generally, R816 was the predominant spot, while Vmm (R390) was absent or indistinguishable from signals seen in some of the negative controls, which is also demonstrated in the blot detected with a pool of all disease sera. There were no detectable false-positive signals due to the His6–ABP tag; however, a weak reaction to the E. coli lysate was seen for some sera. The five control sera from healthy bovines and FBS showed that there is no general cross-reactivity of serum IgGs to the recombinant proteins. A faint positive signal, considered to be noise, did occur for four of the control sera.
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. The protein-specific signals were significantly reduced in the pre-adsorbed sera compared to untreated sera, while all other proteins acted as controls and reacted with equal signal intensities, indicating specific binding of the serum antibodies to the recombinant proteins. Since a more concentrated PG1T lysate was used, the PG1T signal is stronger in comparison to Fig. 1
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and all results are compiled in Table 3. The His6–ABP-antibody staining showed clear and strong bands at the expected sizes of the recombinant proteins and for the His6–ABP control. Protein R117 had a second weak band of smaller size, indicating partially degraded protein or proteolytic fragments, which was further substantiated by the serum-detected blots that also showed bands of smaller sizes. A comparison of His6–ABP and Ponceau S stains showed that only small amounts of contaminating proteins were present, although protein R364 had several bands corresponding to E. coli proteins. There were no interactions with the His6–ABP tag in any of the Western blots. All five recombinant Vmm-type proteins generated bands with the CBPP sera but with different signal intensities and with variations between sera. The previously studied Vmm was the least immunoreactive protein, generating weak and therefore inconclusive bands with sera from three natural infections and a distinct band only in the experimental infection. Despite the small quantity of R816 on the membranes, it generated the strongest signal with most sera. It is noteworthy that the staining of the M. mycoides SC lysate varied among the sera even with identical aliquots of the same lysate. Blots with the control sera from healthy bovines and secondary antibodies were blank or showed rare, extremely faint bands; hence, the noise levels in the disease serum blots were considered to be negligible.
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Generation of Vmm-type protein-specific antibodies
pAbs to the five recombinant proteins were generated in rabbits. In addition, the mouse mAb 5G1, which targets Vmm (Persson et al., 2002), was used. The rabbit sera were affinity-purified using the recombinant proteins as ligands to obtain monospecific polyclonal antisera. The cross-reactivity of the polyclonal antisera was tested by dot blotting in duplicates. The six recombinant proteins, an M. mycoides SC strain PG1T lysate and four recombinant proteins of human origin were analysed with the five affinity-purified pAbs and the 5G1 mAb (Fig. 4). All five antibodies against the recombinant proteins generated signals with their respective partner at 1 : 10 000 dilutions. A117, A816 and A847 generated weak cross-reactivity with each other. None of the specific pAbs stained the PG1T lysate. The 5G1 staining showed strong specific staining of the recombinant Vmm (R390) and weak staining of the PG1T lysate. Furthermore, there was no cross-reactivity with the His6–ABP tag or the recombinant proteins of human origin produced using the same vector and methodology, which were used as negative controls. It is noteworthy that antibodies A816 and A1033 will probably bind all the homologues to proteins MSC_0816 and MSC_1033 mentioned in the ‘Selection and in silico analysis of Vmm-type proteins’ section above.
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), which confirms the bioinformatic prediction that protein MSC_0364 has variable expression. Approximately 20 % of the colonies in M223/90 were stained, but only 5 % in PG1T. Antibody A847 gave an inconclusive result in that one colony was positive among thousands of negatives in strain M223/90 only; however, this may be a result of very rare expression of MSC_0847 under in vitro conditions. Expression of MSC_0117, MSC_0816 and MSC_1033 could not be detected with antibodies A117, A816 or A1033 by colony blotting. The control experiment with mAb 5G1 showed a high frequency of variation in the expression of Vmm in PG1T, in accordance with previous results (Persson et al., 2002).
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). Antibody A364 generated a strong band of the correct size and two smaller bands that were considered to be noise, thus supporting the previous colony blotting results. Antibodies A117 and A847 gave a single band that would support the hypothesis that MSC_0117 and MSC_0847 were expressed, but the protein sizes were different from the theoretical sizes of the native proteins (see Table 2). Two distinct protein bands were obtained with A1033, indicating that both MSC_1033 and its homologue MSC_1058 were expressed. Even though the lower band corresponds to the theoretical size of MSC_1058 (15.7 kDa), it is tempting to assume that both proteins have retarded migration. No bands were obtained for antibody A816 despite the fact that this antibody should recognize five protein homologues. It is noteworthy that these Western blots contained a more concentrated PG1T lysate than the dot blots.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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When sera from natural CBPP infections were analysed for the presence of antibodies that target Vmm-type proteins, it was found that all sera contained antibodies to some or all of the Vmm-type proteins or their homologues. However, the sera generated different dot and Western blot patterns (Figs 1 and 3, Table 3), suggesting that the immune responses in the individual bovines were triggered by different proteins, either because this is the nature of the immune system or because the protein composition on the mycoplasma surface differed due to variable expression of each Vmm-type protein. For most sera, dot and Western blotting gave similar results, but we could observe differences in signal strength. This is expected due to the conformational differences of the proteins in the assays. Surprisingly, the previously studied Vmm (MSC_0390), which is thought to be immunodominant, generated the poorest IgG response and no response at all in most sera. Protein MSC_0816 and its homologues generated the strongest response overall. Interestingly, the differences in immunoreactivity between individual sera from one outbreak were as pronounced as the deviations seen between sera from different outbreaks in vastly separated geographical regions. This is clearly seen with the Botswana serum G2, which has a particular protein profile, whereas most Namibian and Botswana sera follow a more uniform pattern. The sera from Tanzania and Kenya gave distinct protein profiles; however, more sera must be analysed to find out if this is representative of the region or just specific for the individual bovines.
The dot and Western blotting results presented were repeated three times and twice, respectively, with high reproducibility. Standardized experimental conditions were used for each set of blots, with identical protein batches and volumes, and fixed serum dilutions. Signal intensities in Table 3 were annotated by judging all available replicates of the blots. It is not possible, however, to directly quantify the amounts of bound IgG antibodies by measuring the signal intensity when using an enhanced-chemiluminescence detection system, since subtle changes will influence the signal strength, such as the amount of protein in the blot, the time for photo acquisition and the enzymic luminescence reaction. Clearly the light emitted from a strong positive signal will suppress the detection of a weak signal on the same blot. An example is the dot blot with serum PW227 in Fig. 1, where responses to the recombinant proteins are weak compared to the PG1T lysate response, which is why the PG1T signal appears more intense than for blots in which one of the recombinants gives the predominant signal. Similarly, a negative control often generated a weak signal when no positive signal was present. For this reason signals were categorized as inconclusive when only weak signals were obtained compared to the controls, or if the higher dilution of a serum lacked the corresponding signal of a lower dilution. Furthermore, we do not know the effects of storage, transport, cycles of freezing and thawing, age, lyophilization, etc. on the immunoglobulins. Naturally, the quality of the disease sera will affect the general signal strength in an experiment, but should have less effect on the relative amounts of protein-specific antibodies that cause the protein profile within one blotting membrane. The only fresh samples used in this study were from the five healthy control cattle, and it is possible that these five samples had a higher general IgG titre than those of the stored CBPP-positive sera.
Having concluded that the Vmm-type proteins or their homologues are expressed in natural infections and generate a humoral immune response, variable protein expression in vitro was also examined. All results for MSC_0364 were congruent and showed that this protein is expressed in a culture medium environment, and the colony immunostaining confirmed that its expression is variable. Transcripts of all Vmm-type proteins were identified by RT-PCR in total RNA of strain PG1T, although this method is very sensitive and does not indicate transcription levels. Colony blots were essentially negative and therefore inconclusive. Western blots of PG1T lysates provided some support for expression of the Vmm-type proteins, with one distinct band for A117 and A847, and two distinct bands for A1033, as expected. There is still some uncertainty regarding these results, since the detected bands were of sizes that differed from the calculated theoretical protein sizes. It is well known, however, that proteins and especially lipoproteins can display unpredictable migration in SDS-PAGE (Banker & Cotman, 1972; Miyake et al., 1978; Simons & Helenius, 1970). At this point one can only speculate whether the failure to detect proteins MSC_0117, MSC_847 and MSC_1033 in colony blots is due to epitope masking or a protein conformation problem that makes the antibodies unsuitable for assays with native, folded proteins, even if they worked well in Western blot applications and dot blotting of recombinant proteins. Using these techniques we were unable to determine if these three proteins are variably expressed. The last protein, MSC_0816, and its homologues could not be detected using any of the immunoassays.
An interesting aspect of the analyses was the deviation between our experimental observations and the expected expression profile of the Vmm-type proteins, as judged from the promoter sequences. Looking at the genomic sequence of M. mycoides SC strain PG1T, most of these Vmm-type proteins would be expected to be silent in this strain according to the length of the promoter spacer. Only one of the expressed Vmm-type proteins, MSC_117, had a promoter spacer length of 17 bp and should theoretically be expressed. Colony blotting indicated that MSC_364 and MSC_390 were expressed, while MSC_117 was not. One explanation would be that the genome sequence and the colony blots were made from different passages of PG1T. Furthermore, transcripts were detected for all the Vmm-type proteins, but the sensitivity of PCR would detect a very small fraction of expressed genes that may be undetectable with the other methods. Generally, comparisons between experiments and batches of cultures are unreliable when working with variable proteins.
It may be argued that the antigen repertoire of in vitro-cultured strains of M. mycoides SC differs from those of strains in natural infections, since vaccination with non-viable in vitro cultivated strains often gives insufficient protection. Therefore, one hypothesis is that vaccine candidates should be searched for within the set of proteins that are expressed in the infected host but not in the laboratory. Theoretically, a vaccine of lysed whole cells spiked with a blend of all Vmm-type proteins should match all expression combinations in infected hosts.
Following the systematic analysis of surface proteins by recombinant technology we have presented results for six Vmm-type proteins that generated immune responses in vivo. We have also shown that the IgG response targets different Vmm-type proteins in individual sera. Further work using serial bleeds during a natural infection would increase our understanding of the natural variability in the expression of these proteins and possible infection-stage-dependent variability. It is appealing to consider these Vmm-type proteins as potential components of a recombinant protein vaccine, although their use in a vaccine needs to be further evaluated, since a humoral immune response does not necessarily mean that these proteins raise a cell-mediated immune response or a protective immune response. A recombinant vaccine to M. mycoides SC would have several advantages over live attenuated vaccines, which include durability in storage and transport, fewer undesirable side effects, since it would be more defined, and most importantly in this case, there are more options to modulate a recombinant vaccine. For example, one can readily add or remove fusion partners to enhance host responses or make di- and multimers of the recombinant proteins in the vaccine.
The recombinant proteins and their corresponding specific antibodies produced in this study have the potential to be powerful reagents for future protein investigations such as ELISA development, immunohistochemistry and studies of protein interactions.
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ACKNOWLEDGEMENTS |
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Edited by: C. Citti
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Received 18 June 2007;
revised 1 November 2007;
accepted 8 November 2007.
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Represents the predicted cleavage site for the prolipoprotein signal peptidase.
