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Clinical and Diagnostic Laboratory Immunology, July 2000, p. 600-606, Vol. 7, No. 4
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning and Expression of Immunoreactive Antigens
from Mycobacterium tuberculosis
Renee Lay
Hong
Lim,1,*
Li
Kiang
Tan,1
Wai
Fun Lau,1
Maxey
Ching Ming
,
Chung,1,2
Roseanne
Dunn,1,
Heng
Phon
Too,2 and
Lily
Chan1
Bioprocessing Technology
Centre1 and Department of
Biochemistry,2 The National University of
Singapore, Singapore
Received 1 December 1999/Returned for modification 8 February
2000/Accepted 4 April 2000
 |
ABSTRACT |
Four immunoreactive proteins, B.4, B.6, B.10, and B.M, with
molecular weights ranging from 16,000 to 58,000, were observed from
immunoblots of Mycobacterium tuberculosis total lysates
screened with sera from individuals with active tuberculosis. These
proteins were identified from microsequence analyses, and genes of
proteins with the highest homology were PCR amplified and cloned into
the pQE30 vector for expression studies. In addition, a 37.5-kDa
protein, designated C17, was identified from a phage expression library of M. tuberculosis genomic DNA. Preliminary immunoblot
assays indicated that these five resultant recombinant proteins could detect antibodies in individuals with active pulmonary and
extrapulmonary tuberculosis. The overall ranges of sensitivities,
specificities, positive predictive values, and negative predictive
values for the recombinant antigens were 20 to 58, 88 to 100, 69 to
100, and 56 to 71%, respectively. The B.6 antigen showed preferential reactivity to antibodies in pulmonary compared to nonpulmonary tuberculosis serum specimens. All of these recombinant antigens demonstrated potential for serodiagnosis of tuberculosis.
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INTRODUCTION |
Tuberculosis (TB) is a major health
problem in the developing world as well as a disease which is
reemerging as a major health threat in the developed world
(6). World Health Organization (WHO) statistics indicate
that one-third of the world's population is currently infected
(34). TB is rated the second most common infectious disease
and has the highest mortality rate of any infectious disease in the
world. Currently there are 30 million cases of active TB worldwide,
with approximately 8 million new cases and 3 million deaths reported
annually. In addition, 50 million people may already be infected with
multidrug-resistant (MDR) strains of Mycobacterium
tuberculosis. A high prevalence of TB is also associated with
human immunodeficiency virus (HIV) infection and AIDS and is now
becoming the leading cause of death among HIV-positive individuals,
with a fatality rate of 80% (34).
Control of this disease revolves around good patient care and
management. In particular, early detection and treatment of tuberculosis can limit transmission of the bacilli. Conventional tests
for the diagnosis of tuberculosis include chest X-ray, direct sputum
smear for acid-fast bacilli, culture test, and the skin tuberculin PPD
(purified protein derivative) test (29). Among these, the
culture method is time-consuming but reliable. PCR and nucleic
acid-based methods for detecting M. tuberculosis DNA sequences require complex equipment and highly skilled staff, and they
are expensive and unsuitable for routine diagnostic testing in
developing countries (5). Rapid serological diagnostic tests such as the enzyme-linked immunosorbent assay (ELISA) and membrane chromatography tests, in contrast, are simple and inexpensive, and the
latter can be point-of-care devices (3). A major problem encountered in serological techniques is the specificity and reactivity of antigens used. A majority of M. tuberculosis antigens
studied to date have homology with analogous proteins of environmental mycobacteria or other bacteria, resulting in unspecific reactivity to
antibodies in patients with inactive TB or nontuberculous infections (8, 23). Hence, positive test results produced by these
known antigens are generally unreliable, and supplementary tests are required to confirm tuberculosis infection. It was shown that the use
of recombinant M. tuberculosis antigens of specific purity or particular epitopes may enhance the specificity and sensitivity of
serological testing for TB when used in a panel of recombinant antigens
(1). Rapid diagnostic tests that are specific and sensitive
would be useful in both seroepidemiological and clinical studies
pertaining to tuberculosis control and prevention. In this report, we
describe the identification, isolation, and characterization of five
recombinant antigens from M. tuberculosis for use as
serodiagnostic markers for tuberculosis.
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MATERIALS AND METHODS |
M. tuberculosis total-protein extraction.
M.
tuberculosis cells (ATCC 27294) were cultured in MycoFlasks
(Gibco, BRL) containing Lowenstein-Jensen medium at 37°C with 10%
CO2 in a humidified incubation chamber (Jouan IG/50 model). Confluent cells from six culture flasks were harvested by adding 3 ml
of Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) into
each flask and gently flushing the surface of the flask. Dislocated
cells were placed into sterile plastic tubes (Falcon), and cells were
pelleted by centrifugation at 1,100 × g for 5 min. Cells
were washed once in an equal volume of distilled H2O before being resuspended in an equal volume of distilled H2O. The
cell suspension was then heated to 90°C for 2 h and frozen at
20°C overnight. Cells were then thawed on ice and pelleted by
centrifugation at 20,000 × g for 10 min. Extraction of
total protein was performed by adding 500 µl of 8 M urea solution to
0.5 g of cell pellet, vortexing the cell suspension at room
temperature for 20 min, and heating it at 90°C for 2 min. Insoluble
cellular debris was removed by centrifugation at 20,000 × g
for 10 min, and the supernatant containing the extracted total protein
was kept at 20°C until further use.
Western blot analysis.
The total-protein extract of M. tuberculosis was fractionated on a sodium dodecyl sulfate-7.5%
polyacrylamide gel electrophoresis (SDS-7.5% PAGE) gel
(20) and transferred onto a nitrocellulose membrane by
Western blotting (33). Strips from the immunoblot were
probed against pooled positive (tested positive by skin PPD and
culture) and negative sera from nine individuals with active TB and
seven healthy individuals, respectively. Incubation with pooled sera
(1:100 in 1% skim milk-TBST [10 mM Tris, pH 7.5, 300 mM NaCl, 0.005%
Tween 20]) was carried out with rocking for 1 h at room
temperature. The blots were then washed four times in TBST before
incubation with alkaline phosphatase-conjugated goat anti-human
immunoglobulin (Ig) (Harlan Sera Lab, Loughborough, United Kingdom)
(1:1000 in 1% skim milk-TBST) for another 1 h. The strips were
again washed four times in TBST followed by incubation in 1 ml of
bromochloroindolyl phosphate-nitroblue tetrazolium substrate (NBT-BCIP;
Bio-Rad) for 4 min. The reaction was stopped by washing four times in
distilled H2O.
N-terminal sequencing.
Individual protein bands (which were
shown to react positively in the immunoscreening experiment) were
excised from several preparative SDS-7.5% polyacrylamide gels and
concentrated by reelectrophoresis (constant current of 18 mA at 8°C)
on a long stacking gel (7 cm of 4% stacking gel, 5 cm of 10%
resolving gel). The concentrated protein bands were blotted onto a
polyvinylidene difluoride (PVDF) membrane (Bio-Rad), stained with
Coomassie brilliant blue R-250 (Sigma, St. Louis, Mo.), and excised for
N-terminal microsequencing. The protein bands were also blotted onto
Hybond-C nitrocellulose membranes (Amersham Life Science, Little
Chalfont, United Kingdom) for validation by immunoscreening using the
same pooled sera samples as described above.
Screening of a phage expression library.
An expression
library of EcoRI-restricted genomic DNA of M. tuberculosis was constructed in lambda ZAP Phage expression
vector, according to the protocol by Stratagene (ZAP Express cDNA
Synthesis kit manual, Stratagene Cloning Systems, 1998). The resultant
library has 98% recombinants (2 × 106 PFU/µg arms)
and insert sizes ranging from 0.7 to 2 kb. A lawn of XL1-MRF' host
cells infected with about 2 × 104 PFU of the phage
stock was prepared on a 150-mm plate and incubated for 6 to 7 h at
42°C. The lawn was then overlaid with a Hybond-C nitrocellulose
membrane (Amersham) presoaked in 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) for induction
of protein expression by further incubation at 37°C for 4 h. The
plate and membrane were indexed for matching corresponding plate and
membrane position. Approximately 0.4 × 106 plaques
were screened, and as a negative control, a lawn of host cells infected
with 2 × 104 PFU of the nonrecombinant lambda ZAP
phage was used instead.
After transfer, membranes were washed twice in TBST buffer and blocked
in 5% skim milk-TBST. Pooled sera from four individuals with active TB
and from four healthy individuals were preabsorbed overnight, against
negative-control membranes. Membranes from the expression library were
incubated with the preabsorbed human sera for 2 h with rocking at
room temperature, followed by three washes in TBST. A secondary
antibody of alkaline phosphatase-conjugated goat anti-human Ig (diluted
1:1,000 1% skim milk-TBST) was added to the membranes and allowed to
incubate for 1 h. After a final wash, colorimetric detection was
performed using NBT-BCIP substrate as described previously. Positive
plaques were cored out, and recombinant phage was eluted in SM buffer
containing 2% chloroform (Stratagene manual). These were replated at
about 100 to 200 PFU on 82-mm plates for secondary and tertiary
screenings using the same preabsorbed pooled sera. Positive recombinant
phage clones from tertiary screenings were subjected to pBlueScript SK
plasmid excision using helper phage, and recombinant plasmids were DNA sequenced using the forward T3 primer (Stratagene manual).
Cloning and expression of M. tuberculosis
antigens.
Synthetic oligonucleotides were designed for PCR and
cloning of the B.4, B.6, B.10, B.M, and C17 genes, including a known 38-kDa antigen (2). M. tuberculosis genomic DNA
was extracted as previously described with a few modifications
(7). The PCR mixture contained 0.1 µg of M. tuberculosis genomic DNA, 50 pmol of synthetic oligonucleotides
(Genset, Singapore), 10 µl of GC-melt (from the Advantage-GC Genomic
PCR kit [Clontech, Inc., Palo Alto, Calif.], 5 µl of 10× Expand
High-Fidelity DNA Polymerase buffer (containing MgCl2), 0.2 mM deoxynucleoside triphosphates (dNTPs), 0.75 U of Expand
High-Fidelity DNA Polymerase (Boehringer GmbH, Mannheim, Germany), and
distilled H2O to a total reaction volume of 50 µl. PCR
amplications were carried out in a DNA PTC-100 thermal cycler (MJ
Research, Watertown, Mass.) under the following conditions: 94°C for
2 min; 9 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 1 min; 19 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 1 min plus 20 s per cycle; and a final extension
step at 72°C for 7 min.
PCR products were initially cloned into pGEM-T Easy vector (Promega,
Madison, Wis.) and subsequently subcloned into pQE30
expression vector
(Qiagen, Inc., Valencia, Calif.). DH5
and Epicurian
Coli XL10-Gold
(Stratagene)
E. coli cells were used for cloning
and
maintenance of plasmid DNA vector and recombinant plasmid
DNA, whereas
M
15 E. coli cells (Qiagen) were used for
expression
studies. Transformation of plasmid DNA into
E. coli cells was
done by a heat shock method as described by Crouse
et al. (
13).
Plasmid DNA extraction was performed using the
Wizard Midi and
Miniprep DNA purification systems (Promega). DNA
sequencing was
carried out on an Automated DNA Sequencer, model 373A
(Perkin-Elmer
Applied Biosystems, Foster City, Calif.) using the
forward (5'-GAATTCATTAAAGAGGAGAAA-3')
and reverse
(5'-GTTCTGAGGTCATTACTGG-3') primers of the pQE30 vector
and
primers synthesized based on internal sequences obtained from
DNA
sequencing of the TB genes. Recombinant proteins were expressed
as
NH
2-terminally polyhistidine-tagged fusion proteins and
purified
from the M
15 E. coli cell extracts to
near-homogeneity by Ni-nitrilotriacetic
acid (NTA) affinity
chromatography (Qiagen, The QIAexpressionist,
3rd ed., July 1997).
Quantitation of recombinant proteins was
carried out using the
DC Protein Assay Kit (Bio-Rad). SDS-PAGE
gels for
visualization were stained either with Coomassie brilliant
blue R-250
or with silver stain (
27).
SDS-PAGE and Western blotting of M. tuberculosis
recombinant antigens.
To maintain consistency, Tris-HCl
two-dimensional (2D) preparative ready gels (Bio-Rad) were used for
Western blotting. A total of 10 µg of purified recombinant antigen
was subjected to SDS-PAGE and Western blotted onto Hybond-C
nitrocellulose membranes (Amersham) using the Bio-Rad TransBlotter
(according to the manufacturer's protocol). After transfer, the
membrane was blocked in 5% skim milk-TBST, air dried, and stored at
4°C until further use.
Immunoblot analysis of recombinant antigens.
Each membrane
containing Western-blotted antigen was cut into strips of 3-mm width (a
total of 23 strips from each blot), and each strip was used for
screening with a serum specimen. One strip was used as an internal
positive control probed with a positive serum specimen that is reactive
to the recombinant protein antigens. A second strip was probed with the
commercially available anti-RGS His probe (Qiagen). Screening was
carried out by incubating each strip in trays with 1 ml of diluted
serum specimen (1:100 in 1% skim milk-TBST) per well for 1 h with
rocking at room temperature. The strips were then washed four times in
TBST, followed by incubation with alkaline phosphatase-conjugated goat
anti-human Ig (Harlan Sera Lab) for 1 h with rocking at room
temperature. The strips were again washed four times in TBST and then
allowed to develop in 1 ml of NBT-BCIP substrate (Bio-Rad) for 4 min.
The reaction was stopped by washing four times in distilled
H2O.
Western blot score.
The reactivities of recombinant proteins
to serum specimens were interpreted based on the intensities of bands
obtained on an X-Rite 400 densitometer (X-Rite Inc.,
Grandville, Mich.). The cutoff values (expressed as densities) for each
individual recombinant antigen were determined based on the range
observed for normal serum specimens.
Serum specimens.
A total of 139 human sera were used in this
study, of which 119 serum specimens were purchased from BioClinical
Partners, Inc., Franklin, Mass., whereas 20 were donated by healthy
laboratory workers. The control groups consisted of two panels, (i) a
panel of 50 serum specimens from healthy individuals, of whom 20 (laboratory workers) had been Mycobacterium bovis BCG
vaccinated previously and 30 (BioClinical) had unknown BCG status, and
(ii) a panel of 19 serum specimens (BioClinical) from individuals with
non-TB respiratory disease (lung cancer).
The test group consisted of 48 serum specimens (BioClinical) from
bacteriologically confirmed active TB patients and 22 serum
specimens
(BioClinical) from patients with inactive TB. The active-TB
serum
specimens comprised 28 from pulmonary-TB and 20 from extrapulmonary-TB
patients. Serum specimens from the inactive-TB panel were from
patients
with positive PPD skin tests but negative acid-fast stains
of sputum
and bacterial culture. All sera were aliquoted and stored
at
70°C
before
use.
Databases and software.
Nucleotide and protein sequence
analysis was carried out using the basic BLAST 2.0 search program from
the National Center for Biotechnology Information (NBCI) and the
M. tuberculosis BLAST server at the Sanger Centre
(Cambridge, United Kingdom).
Statistical analysis.
Sensitivities, specificities, and
positive and negative predictive values were calculated using the Win
Episcope 1.0 (Borland International Inc.).
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RESULTS |
Identification and isolation of M. tuberculosis
antigens.
Immunoblot analysis of M. tuberculosis total
proteins revealed protein bands which reacted with the pooled active
sera but not with the pooled normal sera. When the respective bands
were concentrated on a long stacking gel, excised, and Western blotted, these bands were reactive with the pooled active sera but not with
pooled normal sera, thus confirming the authenticity of these excised
proteins as those initially observed in the primary screening (Fig.
1). These proteins were identified by
homology searches against protein sequence databases, which gave a high
percentage of homology to Mycobacterium proteins (Table
1). PCR primers were designed to isolate
and clone the genes coding for four proteins (B.4, B.6, B.10, and B.M)
which gave the highest matches based on a BLAST homology search against
the SwissProt database.
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FIG. 1.
(I) Gel-purified and concentrated M. tuberculosis protein antigens (1, 2, 3, 4, 5, 6, 7,
8, and 9) blotted onto PVDF membranes were
excised for N-terminal sequencing. (II) These protein antigens were
blotted onto nitrocellulose membranes and immunoscreened against pooled
normal (N) and active-TB (A) sera, respectively. Positive bands
(arrows) were observed with A but not with N.
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Concurrently, primary screening of the phage expression library gave
eight reactive phage recombinants, of which six were
further confirmed
positive by secondary and tertiary screenings
(Fig.
2). These clones were subjected to
plasmid DNA excision,
and restriction enzyme digestions with
EcoRI indicated that all
the clones contained a 2-kb insert.
DNA sequencing revealed that
all the clones were identical, having a
1.161-kb open reading
frame (in frame with the vector's ATG initiation
codon) which
coded for a proline-rich protein. A summary of all five TB
antigens,
with the respective gene sizes, theoretical molecular
masses,
and pI values is shown in Table
2.
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FIG. 2.
Screening of the lambda ZAP Phage expression library of
EcoRI-restricted genomic DNA of M. tuberculosis,
using pooled human sera from individuals with active TB. (A) Primary
screening; (B and C) secondary screening; (D) negative control
consisting of plaques of nonrecombinant phages. Arrows indicate plaques
containing positive recombinant phage.
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Expression and purification of recombinant antigens.
Expression was detected by probing immunoblots containing these
antigens using the commercial anti-RGS His antibody. The levels of
expression observed were high for the B.4, B.M, and 38-kDa proteins,
moderate for the B.6 and B.10 proteins, and low for the C17 protein
(Fig. 3). All of these recombinant
proteins, except for C17, were present in the insoluble fraction of an
SDS-PAGE analysis (data not shown), indicating that these proteins
formed inclusion bodies and were insoluble. As such, these recombinant proteins were purified by Ni-NTA affinity chromatography in 8 M urea,
and the SDS-PAGE profile of the purified antigens subsequently used for
immunoblots is shown in Fig. 4. The
approximate yields of recombinant antigens purified through Ni-NTA were
36 mg/liter for B.4, 0.5 mg/liter for B.6, 0.2 mg/liter for B.10, 15 mg/liter for B.M, <0.1 mg/liter for C17, and 10 mg/liter for the
38-kDa protein.
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FIG. 3.
Expression and affinity chromatography purification
profiles of the M. tuberculosis antigens expressed in 100 ml
of M15/E. coli cultures. Lanes: M and Ms,
protein molecular weight markers (M, Kaleidoscope standards; Ms, Sigma
Broad range); 1 through 4, aliquots taken at 0, 1, 2, and 3 h,
respectively, after induction with 1 mM IPTG; B, total cell lysate
before passing through Ni-NTA column; A, total lysate after passing
through column; W, wash fractions in 8 M urea buffer (pH 6.5 to 5.9);
E1 to E7, eluted fractions in 8 M urea buffer (pH 4.5). The bulk of the
recombinant proteins were observed to be eluted in fractions in 8 M
urea buffer (pH 4.5). The bulk of the recombinant proteins were
observed to be eluted in fractions E2 and E3 (arrows). All the gels
were stained with Coomassie brilliant blue, except for C17, which was
silver stained.
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FIG. 4.
Recombinant M. tuberculosis antigens (arrows)
purified by Ni-NTA affinity chromatography as observed on an SDS-PAGE
gel (silver stained) (A) and on a Western blot of a duplicate gel which
was probed with anti-RGS His antibody followed by detection with
alkaline phosphatase-conjugated goat anti-mouse Ig and NBT-BCIP
substrate (B). Some truncated products of the recombinant proteins were
observed which were detected by the antibody. Lanes: M, Kaleidoscope
prestained standards; 1, B.4; 2, B.6; 3, B.10; 4, B.M; 5, 38-kDa
protein; 6, C17.
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Reactivities of the recombinant antigens to TB serum
specimens.
The different reactivities of recombinant TB antigens
on immunoblot strips probed with serum specimens and anti-RGS His are shown in Fig. 5. The respective cutoff
values for determining reactivity to the different recombinant antigens
were obtained based on the mean densities observed in sera from the
control group of healthy individuals (n = 50). The
cutoff values were densities of >0.04 for the B.4 and B.6 bands,
0.04 for the B.10 and B.M bands, 0.15 for the C17 band, and >0.15
for the 38-kDa band.
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FIG. 5.
Immunoscreening of recombinant TB antigens by a Western
blot assay. Arrows indicate the positions of recombinant antigens on
the immunoblots. Each strip was probed with different serum specimens.
A strip was probed with anti-RGS His (H) to indicate the position of
protein on the blots. The positive control (+) is represented by a
strip probed with a known serum specimen which is reactive to the
specific recombinant antigen.
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Based on the Western blot assay, the reactivities of these antigens to
a panel of active-TB serum specimens are shown in Table
3. Percentages of reactivities and
positive and negative predictive
values for each antigen were
calculated based on sera from infected
individuals (
n = 48; pulmonary and extrapulmonary TB) and sera
from the control
group of healthy individuals (
n = 50), with a
P value of <0.05. The specificities for the B.4, B.6, B.10,
B.M,
C17, and 38-kDa recombinant antigens are 94, 88, 100, 96, 90,
and
98%, respectively. All of the recombinant TB antigens showed
substantial reactivity to active-TB specimens, both pulmonary
and
extrapulmonary. The B.4 antigen was reactive with 58.3% of
the
active-TB panel, compared to 37.5% detected by the known 38-kDa
antigen. In addition, the B.4 antigen showed reactivity to 27.3%
of
the inactive-TB specimens compared to other TB antigens, which
exhibited lower percentages of reactivity to these specimens (Table
3).
The B.6 antigen was found to exhibit specific reactivity
to
pulmonary-TB specimens (46.4%) compared to extrapulmonary specimens
(5%) (Fig.
6). All the other antigens
were able to detect antibodies
in both pulmonary- and extrapulmonary-TB
specimens.
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TABLE 3.
Reactivities and positive and negative predictive values
of recombinant TB antigens against different sera panels
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FIG. 6.
Graph showing percentages of reactivity for the
recombinant TB antigens against the pulmonary-TB (n = 28) and extrapulmonary-TB (n = 20) serum
specimens. The B.6 antigen detected antibodies in 46.4% of the
pulmonary-TB compared to only 5% of the extrapulmonary-TB serum
specimens. The rest of the TB antigens did not exhibit such significant
differentiation of reactivity between the two serum panels.
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DISCUSSION |
A number of M. tuberculosis antigens have been
identified and characterized by various methods employing polyclonal
antibodies from rabbits or monoclonal antibodies (MAbs) from hybridomas
generated from immunized mice. Such antibodies were used widely for
identification and purification of protein antigens by affinity
chromatography (22), immunoscreening of clones from DNA
libraries of M. tuberculosis (24, 37), and
analyses of total-cell lysates or secretory proteins from culture
medium by both one- and two-dimensional gel electrophoresis (16,
32). Immunogenicity in animals (e.g., mice or rabbits), however,
may not reflect relevance to human immune responses. Thus, attempts
were made to search for candidate serodiagnostic antigens by directly
testing mycobacterial proteins with tuberculous-patient sera on
immunoblots of one-dimensional and two-dimensional separations of
antigenic extracts or culture filtrates of M. tuberculosis
H37Rv (4, 26).
In this study, we used antibodies present in the sera of infected
individuals to screen total-cell lysates and a phage expression library
of M. tuberculosis DNA. To date, there is no single
immunodominant species-specific antigen for detection of tuberculosis.
We have chosen to use pooled sera from several infected individuals to allow identification of several immunoreactive antigens reactive to
antibodies present in each serum. In addition, the M. tuberculosis genome database completed by the Sanger Centre
(12) allowed for the rapid identification of these
immunoreactive antigens by homology searches against available protein
and gene databases, which also facilitated the identification of these
gene sequences for cloning.
We have successfully identified and characterized five antigens using a
Western blot total-cell-lysate approach; of these, the B.6, B.10, and
C17 antigens are novel and showed high degrees of nucleotide identity
to unpublished M. tuberculosis H37Rv genes. Based on DNA
sequencing results, the B.6 antigen gene was found to have 99%
nucleotide identity to a gene coding for a protein with homology to
exported proteases or peptidases. The B.10 antigen exhibited 99 and
98% nucleotide identities to the M. bovis acyl coenzyme A
(CoA) synthase (accession no. U75685) and mycocerosic acid synthase
(accession no. M95808) genes, respectively. The C17 antigen exhibited
99 to 100% nucleotide identity to a gene coding for PE-PGRS
(polymorphic GC-rich repetitive sequence) proteins, a member of PE
(proline-glutamic acid) families of clustered genes coding for
glycine-rich proteins which may have immunological and pathogenic
implications (12).
The B.4 antigen exhibited 99.8% amino acid and 98.9% nucleotide
sequence homology to the Cpn-60 protein reported by Kong et al.
(18). The diagnostic potential of a 65-kDa protein (also a
Cpn-60 family of heat shock proteins) by both serological and PCR
methods has been demonstrated (28). The B.M antigen has 99%
nucleotide identity to the reported M. tuberculosis 14-kDa antigen gene (accession no. M76712) and the gene for the 19-kDa major
membrane protein purified from the virulent Erdman strain of M. tuberculosis (21). The serological value of this 19-kDa antigen was shown by 85% reactivity to a panel of 56 sera from individuals with active pulmonary TB (9).
Antigens of diagnostic importance for M. tuberculosis
identified to date include the 65-, 45-, 30/31-, 19-, and 12-kDa
proteins and the 38-kDa lipoprotein (9, 11, 14, 36). Among
these, the 38-kDa protein was shown to be the most specific and
sensitive for detecting antibodies against M. tuberculosis
and is specific for TB complex species (35). As such, we
have chosen to clone and express this antigen to be used in
immunoscreening against the serum panels for comparison with our
recombinant TB antigens. This 38-kDa antigen was expressed as an
insoluble protein in the pQE30/E. coli system; similarly,
Singh et al. reported the expression of this 38-kDa antigen as an
insoluble unfused protein in E. coli (31).
We have chosen the Qiagen expression system for cloning and expression
of the TB antigens. Each expressed recombinant protein contained a
nonimmunogenic 6× His tag at the N terminus which could be
immunodetected by anti-RGS His. A substantially low expression level
was observed for the C17 protein. This may be due to the codon usage of
this protein, which is rich in proline (46.6%), as it is reported that
the expression level of a gene decreases with an increase in the use of
rare codons (17). In addition, the Kyte-Doolittle hydropathy
plot revealed that it is very hydrophilic, which explains the soluble
nature of this protein (19). As most of the recombinant
antigens were insoluble, we have chosen a Western blot assay for
preliminary screening against serum specimens.
Immunoblot assays using human sera were described previously for
analysis of HPLC-purified 45/47-kDa antigen complex (15). Rovatti et al. reported a semiquantitative Western blot serological test to identify PPD-positive individuals, using a discriminate score
for the M. bovis BCG antigen complex A60 against MAbs
(25). In our immunoblot assay system, we used
affinity-purified recombinant proteins to detect antibodies in serum
specimens and have included the known 38-kDa lipoprotein as a control
for our purification and Western blot assay system. Zhou et al.
reported the use of this antigen in a rapid membrane-based assay that
gave a specificity of 92%, very close to our in-house immunoblot assay
of the 38-kDa protein, which gave 98% specificity (38). The
38-kDa antigen was included in the assay system as a further control
for specificity and sensitivity. This was further compared to a
commercially available diagnostic test kit which uses two antigens, one
of which is the 38-kDa antigen. All 13 of the 18 active-TB serum
specimens that tested positive with the 38-kDa protein in our Western
blot assay also tested positive with the kit. The percentage of
specificity of the 38-kDa antigen in this assay is comparable to that
with the kit (98 and 100%, respectively).
Data analysis from our assay system indicated that the B.4, B.6, B.10,
B.M, C17, and 38-kDa recombinant antigens reacted with antibodies in
serum specimens of TB-infected individuals. The reactivities were
sufficiently differentiated from those of serum specimens from healthy
individuals and from individuals with inactive TB or lung cancer.
Screening of the same serum panels using two commercially available TB
diagnostic kits of high specificity indicated the presence of
immunologically specific antibodies reacting to these TB antigens
(submitted for publication). Reports have also shown that other
recombinant antigens did exhibit low levels of cross-reactivities to
sera from healthy individuals, which may be due to cross-reactive
epitopes or analogues to other bacterial proteins (8).
To circumvent this problem, the use of MAbs with recombinant antigens
in competition with patient sera as a test assay was reported to give a
high degree of specificity (10). Alternatively, we have to
further optimize the immunoblot assay to decrease cross-reactivities.
In conclusion, we have demonstrated the identification and cloning of
five TB antigen genes for expression in an E. coli system and shown the potential of each recombinant antigen as a serodiagnostic marker for detection of TB infections. We are in the process of validating the diagnostic utility of these antigens in various test formats.
| |
ACKNOWLEDGMENTS |
We are grateful to Christopher Froggatt, Kean Chong Loh, Cindy
Luo, and Adrian Lee at the Fermentation and Purification Laboratory of
BTC for generously helping us to produce and purify the recombinant antigens for screenings.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Bioprocessing
Technology Centre, National University of Singapore, 5th Floor, MD11, 10 Kent Ridge Crescent, Singapore 119260. Phone: 65-874-6222. Fax:
65-775 4933. E-mail: btclimr{at}nus.edu.sg.
Present address: Department of Cell and Molecular Biology,
University of Technology, Sydney, NSW, Australia.
| |
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Abstract
Introduction
