Archives of Razi Institute, Vol. , No.

, Summer () - Copyright by
Razi Vaccine & Serum Research Institute

Cloning and sequencing of Toxoplasma gondii major surface

antigen (SAG) gene

Solhjoo, K., Ghafari Far*, F., Dalimi, A., Sharifi, Z.

. Department of Parasitology, Medical Sciences Faculty, University of Tarbiat Modarres, Tehran, Iran
. Research Center of Iranian Blood Transfusion Organization, Tehran, Iran

Received May ; accepted Mar

ABSTRACT
Genetic typing methods of T. gondii strains have been extensively perfected in recent years. From a
technical point of view, many tools usable for genetic studied on single-copy loci have been used: RFLP,
PCR-RFLP, sequencing, RAPD-PCR and isoenzyme analysis. We described the cloning and sequence
analysis of the gene which encodes the major surface antigen (SAG or P) of T. gondii. SAG is the
immunodominant antigen of Toxoplasma gondii tachyzoites being considered as the most promising
molecule for a recombinant vaccine or such as DNA vaccine against toxoplasmosis. In the present work,
first, genomic DNA of Toxoplasma gondii was extracted and used for amplifying of SAG gene as a
template. Then PCR product was cloned into pTZR/T vector and plasmid containing SAG gene (pT-
SAG) was extracted from transformed bacteria and SAG gene cloned into pTZR/T was sequenced.
Results showed that the P gene contains no introns and can extract it from genomic DNA of tachyzoite
stage. Results showed also that SAG gene is cloned in pTZR/T plasmid, forming pT-SAG
recombinant plasmid and E. coli TG strain is the best host for pT-SAG transformation. Sequence
analysis of SAG gene cloned into pTZR/T vector showed that SAG gene sequence from a high
virulent strain of T. gondii (Known as RH strain) has sequence identity with P-Br strain, P strain
and C strain and high homology of with RH strain and ZS strain.

Keywords: Cloning, Sequencing, Toxoplasma gondii, SAG, P

INTRODUCTION of major medical and veterinary importance, being a

cause of congenital disease and abortion in humans
Toxoplasmosis, caused by an intracellular protozoan
and in domestic animals (Bhopale ). There are
parasite, Toxoplasma gondii, is widespread
marked biological differences among Toxoplasma
throughout the world (Bhopale ). The disease is
gondii stocks concerning their pathogenicity to mice:
most of the stocks are avirulant in mice producing
asymptomatic chronic infections, while few which
*
Author for correspondence. E-mail: [email protected] are highly virulent in mice stocks produce acute
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toxoplasmosis killing all mice with less than of Medical sciences Faculty of Tarbiat Modarres
tachyzoites. Isoenzyme analysis using six different University, Known as RH strain), maintained in
enzyme systems allowed the identification of BALB/c mice by serial intraperitoneal inoculation of
zymodemes (Z-Z) among a population of about tachyzoites, was used for production of
stocks (Ajzenberg et al a). For biologic and tachyzoites.
epidemiologic studies, three main genotypes are Genomic DNA extraction. About T.gondii
generally recognized in the T. gondii population: tachyzoites (l) were concentrated by
type I, II, and III (Ajzenberg et al b). Lekutis et centrifugation, washed with phosphate buffer saline
al () believed that in addition to (PBS), then lysed in l lysis buffer (M Tris-
developmentally regulated differences in SAG HCl pH containing sodium dodecyl sulphate,
expression, there is measurable allelic variation M NaCl and lmM EDTA) and then treated with
between the three prototypic strains of T. gondii. l proteinase K (g/ml) at C for hr
Interestingly, just to alleles were identify at the (Kimbita et al ). The lysate was then added to
SAG and SAGA luci when Type I, II and III an equal volume of phenol/chloroform () to
strains were compared. In fact, most SAGs are remove proteins. This mixture was centrifuged at
dimorphic. SAG or P protein has an apparent rpm for min and an equal volume of
M.W. kDa (Kasper et al ) and is stage chloroform was added to the supernatant which was
specific, being detected only in the tachyzoite stage, then re-centrifuged. The supernatant was mixed with
but absent in the sporozoite and bradyzoite stages  volume of M sodium acetate and two volumes
(Burg et al , Hunter et al , Kimbita et al of ethanol to precipitate DNA by
).This Antigen is abundant and homogeneously centrifugation at rpm for min. The DNA
distributed on the surface of both extracellular and pellet was washed with ethanol, dissolved in
intracellular tachyzoites (Burg et al ). SAG has sterile distilled water and stored at -oC until use
two glycoforms (Zienker et al ) and is a highly (Sambrook et al ). DNA extraction products
conformational antigen (Chen et al ). The gene were detected in agarose gel and
encoding SAG occurs as a single copy, without photographed.
introns (Kimbita et al , Biemans et al ) and PCR amplification and gel electrophoresis.
is highly conserved in T.gondii strains (Letscher-Bru Genomic DNA isolated from tachyzoites was used
et al , Burg et al ). We are interested in the as a template to amplify the SAG gene by PCR
role of P in the parasites life cycle. Because of performed in l of solution containing l of
this and its importance in the immune response to T. template DNA, l dNTP, l Taq DNA
gondii infection (and therefore its potential as a polymerase, l X PCR buffer, l MgCl,
diagnostic tool and/or subunit or DNA vaccine), we l distilled water and l each of primers
have studied further molecular characterization of [Forward primer, nt: introduced Hind III
this protein through cloning and sequencing of the P recognition site, underlined: -ATT AAG CTT
gene. ATG TTT CCG AAG GCA GTG- (- nt);
Reverse primer, nt; introduced EcoRI recognition
site, underlined: -ATT GAA TTC TCA CGC
MATERIALS AND METHODS
GAC ACA AGC TG-(- nt)] under the
Production of T. gondii tachyzoites. A high following conditions: After an initial min
virulent strain of T. gondii (presented in denaturation at C, each cycle consisted of s at
experimental laboratory of Parasitology Department C, s at C and s at C at the end of
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the cycles of amplification, a final extension was (Fermentas) to screening of blue and white colonies
continued for min at C. and incubated at C for hr. Several white and
The PCR products analyzed by electrophoresis on blue colonies were randomly selected from each agar
a agarose gel and photographed. The size plate and inoculated in a LB medium containing
markers used to estimate PCR products were the ampicillin and incubated at C for hr.
bp and kbp DNA ladders (Fermentas). The Confirmation of Cloning of SAG gene into
DNA sequence of gene encoding the surface antigen pTZR/T vector. The plasmid was purified from
P (SAG) of T. gondii was obtained from the white and blue colonies of bacteria by Accuprep
GenBank database (http//www.ncbi.com) with plasmid Extraction Kit (BioNEER), following the
accession No. AY and bp. The forward manufacturers protocol. After plasmid extraction,
and reverse primers were designed according to the following experiments were performed for
nucleotide sequence in GenBank database and improving cloning of SAG gene into pTZR/T
GenRuner Software. vector:
Extraction of PCR products (SAG gene band) ) Comparison of extracted plasmids on
from agarose gel. PCR products were purified using agarose gel. l of each plasmid extracted from
a DNA Extraction Kit from agarose gel (Ferments), white (pT-SAG) and blue (pTZR/T) colonies
following the manufacturers recommendations. bacteria were loaded on a agarose gel and were
Ligation of SAG gene to pTZR/T Cloning electrophoresis and photographed. Then, plasmid
vector. The purified PCR products were ligated to bands on agarose gel were compared.
pTZR/T cloning vector (InsT/Aclone PCR ) PCR amplification of SAG gene using by pT-
product cloning kit, Fermentas), following the SAG plasmids. Plasmid DNA extracted form white
manufacturers protocol. colonies bacteria (pT-SAG) was used as a template
First ligation reaction was prepared l of solution, to amplify the SAG gene by PCR performed in
containing l of pTZR/T plasmid, l purified l of solution under condition previously description
PCR products (SAG gene), l T DNA ligase, in part . The PCR product were analyzed by
l lX buffer, l PEG and l Nuclease free D.W. electrophoresis on a agarose gel and
After vortex and spin, this mixture was incubated at photographed. The size marker used to estimate PCR
C for overnight, and then ligation reaction products were the bp and kbp DNA ladders
product was stored at C until use. (Fermentas).
Transformation and Screening. Preparation of ) Enzyme digestion of pT-SAG plasmids. With
competent cells from Escherichia coli TG strain regard to designed HindIII and EcoRI restriction
was performed by calcium chloride method enzymes sites respectively on forward and reverse
(Sambrook et al ). primers and present them in recombinant plasmids
For transformation, l of ligation reaction product extracted from white colonies bacteria (pT-SAG),
was added to l competent cells, after vortex these plasmids were digested by HindIII and EcoRI
and spin the mixture was incubated at C for s, enzymes. For this propose each enzyme digestion
and immediately was placed on ice for -min. The reaction was performed in l of solution
transformed cells were allowed to recover in l containing l plasmid , l restriction enzyme,
LB broth medium free antibiotic by incubated at l X buffer and l D.W, after spin and vortex,
C for - hr with shaking. These recovered cells this mixture was incubated in C for overnight.
were plated onto LB agar plates containing Because of being different of restriction enzyme
ampicillin, IPTG (Fermentas) and X-Gal buffers, each enzyme digestion was performed
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separately. Total of enzyme digestion products by

EcoRI was loaded on a agarose gel and the band
resulting from digestion by EcoRI enzyme
containing SAG fragment was extracted from
agarose gel by DNA extraction Kit from agarose gel
(Fermentas) and second enzyme digestion by HindIII
was performed on it. Products from digestion by
HindIII were loaded on a agarose gel and the
band resulting from digestions by two enzymes was
analyzed by electrophoresis.
) Nucleotide sequencing of the SAG cloned in
pTZR/T vector. The plasmids extracted from
white colonies bacteria (pT-SAG) were sequenced
by Faza Biotech Company.

Figure . Ligation patterns for construction of recombinant

RESULTS pT-SAG plasmid with pTZR/T (cloning vector) and
SAG gene.
DNA extraction. Fig. shows that genomic DNA
has been extracted by using lysis buffer and
proteinase K followed by phenol /chloroform Figure . Genomic DNA
method. extraction from Toxoplasma
gondii tachyzoites was
PCR amplification. Fig. shows that DNA performed by lysis buffer
and phenol : chloroform
fragment PCR amplified was about bp and method and electrophoresed
similar to the Toxoplasma gondii SAG gene size in agarose gel.
and no any genes was amplified exception with
SAG gene. Thus, these designed primers are
specific for amplifying of SAG gene. Results from
electrophoresis of PCR products with extracted
plasmids pT-SAG using specific primers and
remembered conditions showed that a bp

fragment of SAG gene was amplified and this gene
has been cloned into PTZR/T plasmid (Figure ).
Ligation of SAG gene into pTZR/T Cloning
vector. According to the figure , there were two
patterns for ligation of SAG gene into pTZR/T
cloning vector. In pattern , introduced HindIII
recognition site of SAGgene is near to EcoRI
restriction site of pTZR/T cloning vector but in
pattern , introduced HindIII recognition site of
SAGgene is far from EcoRI restriction site of Figure . PCR amplification and gel electrophoresis of PCR
product. Lane , , and : PCR product (approximately bp);
pTZR/T cloning vector. Lane : bp DNA ladder; Lane : Kbp DNA ladder.
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Comparison of extracted plasmids on Comparison of bands of extracted plasmids from

agarose gel. Electrophoresis of extracted plasmids white and blue colonies bacteria shows that bands of
showed that both of plasmids (pTZR/T and pT- plasmids extracted from white colonies are heavier
SAG) had three bands (linear, open circular and than plasmids extracted from blue colonies and thus,
super-coil plasmids respectively from up to down) in SAG gene has been cloned into pTZR/T
which pT-SAG bands placed above of pTZR/T (Figure ).
bands on agarose gel (Figure ). Enzyme digestion. According to the ligation
patterns (figure ), when plasmids extracted from
Figure . Comparison of white colonies are digested by EcoRI restriction
extracted plasmids on
agarose gel showed that both of enzyme, digestion products may have two different
plasmids (pTZR/T and pT- electrophoresis patterns. Figure showed that
SAG) had three bands (open
circular, linear and super-coil electrophoresis of digestion products obtained from
plasmids respectively from up to the first digestion by EcoRI had two bands in that,
down) in which pT-SAG bands
placed above of pTZR/T bands one was less than bp and the other was less
on agarose gel : pTZR/T than bp. Figure showed that when plasmids
(Lane ) and pT-SAG (Lane ).
extracted from white colonies were digested by
EcoRI restriction enzyme, one band (approximately
less than bp) was observed. In both above sets
of enzyme digestion, electrophoresis of digestion
products obtained from the second digestion by
HindIII (that performed on digestion products
obtained from the first digestion by EcoRI) showed a
bp band (Figure and ). Figure showed
that second digestion by HindIII on pT-SAG
digested by EcoRI, had two bands in that, one was
bp (down) and the other was less than bp
(up). Results from enzyme digestion revealed that if
the plasmid extracted from white colonies bacteria
(pT-SAG) were digested with EcoRI and HindIII, a
A B
Figure A. Agarose gel electrophoresis of Digestion of bp band was cut and separated that this is SAG
extracted pT-SAG (according to ligation pattern ) after gene, and thus the SAG gene has been cloned into
transformation; Lane : Kbp DNA ladder; Lane : pT-SAG
has three bands (linear, open circular and super-coil plasmids pTZR/T.
respectively from up to down); Lane : pT-SAG digested by
EcoRI has two bands in that, one is less than bp (down)
and the other is less than bp (up); Lane : Second Figure . Agarose gel
digestion by HindIII on less than bp band (containing
electrophoresis of PCR
SAG); Lane : bp DNA ladder. amplification of SAG
gene products with pT-
Figure B. Agarose gel electrophoresis of Digestion of SAG and Genomic DNA;
extracted pT-SAG (according to Ligation pattern ) after Lane : Kbp DNA ladder;
transformation; Lane : Kbp DNA ladder; Lane : pT-SAG Lane : PCR amplification
has three bands (linear, open circular and super-coil plasmids of SAG gene with pT-
respectively from up to down); Lane : pT-SAG digested by SAG; Lane : PCR
EcoRI has one band (approximately less than bp); Lane : amplification of SAG
Second digestion by HindIII on pT-SAG digested by EcoRI, gene with Genomic DNA;
has two bands in that, one is bp (down) and the other is less Lane : bp DNA
than bp (up); Lane : bp DNA ladder. ladder.
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Nucleotide Sequencing. Nucleotide sequence Results also showed that according to the ligation
analysis of the SAG cloned in pTZR/T vector patterns (figure ); digestion products may have
revealed sequence identity with P-Br strain two different electrophoresis patterns (figure
(GenBank Accession No. AY), P strain and ) and different ligation patterns have no
(GenBank Accession No. S), and C-strain effect on cloning and sequencing.
(GenBank Accession No.S). Sequence Sequence analysis of SAG gene cloned into
analysis of the SAG region revealed high homology pTZR/T shows that the sequence has
of with RH strain (GenBank Accession No. identity with P-Br strain, P strain and C Strain and
AY) and ZS Strain (GenBank Accession high homology of with RH strain and ZS
No. S). strain. This result shows that SAG dimorphism
and chromosomal localization are windows
through which are population biology of T. gondii
DISCUSSION can be observed and is similar to reports of other
Genetic typing methods of T. gondii strains have researchers (Letscher-Bru et al , Burg et al
been extensively perfected in recent years. From a ) about highly conserved of SAG sequence
technical point of view, many tools usable for in T. gondii strains and Lekutis et al () that
genetic studied on single-copy loci have been believed that in addition to developmentally
used: RFLP, PCR-RFLP, sequencing, random regulated differences in SAG expression, there is
amplified polymorphic DNA PCR (RAPD-PCR) measurable allelic variation between the three
and isoenzyme analysis. Most of these studies prototypic strains of T. gondii. Interestingly, just
were performed on a small sampling of stocks and to alleles were identify at the SAG and SAGA
described the use of only one locus, mainly SAG luci when Type I, II and III strains were compared.
locus, for genetic typing (Ajzenberg et al b). In fact, most SAGs are dimorphic.
We describe the cloning and sequence analysis of Burg et al () showed that comparison of near-
the gene which encodes the major surface antigen full length cDNA to genomic DNA by sequence
(SAG or P) of T. gondii. Results showed that and restriction mapping (as well as full length
the P gene is a single copy, contains no introns protection of the end of P mRNA with
and can extract it from genomic DNA of genomic DNA) demonstrate that the P gene
tachyzoite stage. Results also showed that SAG contains no introns and northern blot analysis
gene is cloned in PTZR/T plasmid, forming pT- shows that the P mRNA is about
SAG recombinant plasmid and E.coli TG strain nucleotides in length and accumulates to very high
is the best host for pT-SAG transformation. levels and predicted size for P primary
Burg et al (), Hunter et al () and Kimbita translation product deduced from the cDNA
et al () also showed that SAG is stage sequence is kDa also showed that there are
specific, being detected only in the tachyzoite two potential methionines for SAG; although
stage, but absent in the sporozoite and bradyzoite translational machinery most often utilizes the
stages and this antigen is abundant and methionine codon it encounters, some data suggest
homogeneously distributed on the surface of both that the second methionine codon of P is used to
extracellular and intracellular tachyzoites. Kimbita initiate translation. Since a signal sequence of
et al () and Biemans et al () also resulted amino acids is unprecedented, this potential signal
that the gene encoding SAG occurs as a single sequence cleavage site would also suggest the
copy, without introns. second methionine codon as the initiator of the
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primary translation product (with a signal peptide Hunter, S.A., Shbaugh, L., Hiar, P., Bozic, C.M. and
of amino acids). Therefore, the amplified Milhausen, M. (). Baculovirus-directed expression
and secretion of a truncated version of Toxoplasma
bp DNA segment in this work utilizes the second
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methionine codon of P and it is used to initiate
-.
translation. Kasper, L.H., Currie, K.M. and Bradley, M.S. (). An
unexpected response to vaccination with a purified
major membrane tachyzoite antigen (P) of
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