Abstract: In the present study we have isolated Toxoplasma tachyzoites from infected mice, RNA was extracted and RT-PCR reaction was conducted to obtain the Toxoplasma gondii heat shock protein 90 cDNA and gene was amplified using PCR. A 2126 bp fragment as PCR product was cloned in pGEMEX-1 expression vector and confirmed by restriction analysis.
INTRODUCTION
Toxoplasma gondii is a ubiquitous obligate intracellular protozoan parasite, which distributes via host circulation and causes some cell defects. A hallmark of the asexual cycle is an interconversion between the rapidly dividing tachyzoite and the more slowly dividing bradyzoite, which is essential for disease propagation and causation. Tachyzoites are responsible for acute illness and congenital neurological disease, while bradyzoites can remain latent within the tissues for many years, representing a threat to immunocompromised patients (Markel et al., 1996).
Acquired toxoplasmosis is a benign disease which usually doses not any symptoms, while congenital toxoplasmosis is a malignant disease (Markel et al., 1996). Intra uterine infection varies based on pregnancy trimester. The risk of congenital disease is low (10 to 25%) when maternal infection occurs during the first pregnancy trimester and goes high (60 to 90%) during the third pregnancy trimester. However, congenital disease is more fatal when infection is acquired in the first trimester (Jones et al., 2003).
The induction of bradyzoite development in vitro has been associated with changes in temperature, pH and other stress inducers known also to activate the expression of heat shock proteins (Tomavo et al., 1991). Toxoplasma tachyzoites are release from host cell at acute phase of infection and invade to uninfected cells. The tachyzoite excrete an 82 kDa protein named heat shock protein 90 (Hsp90) to facilitating their invasion (Ahn et al., 2003). It is a potential drug target and inhibition of this protein may protect of infection (Echeverria et al., 2005).
Since there are not enough informations about the Hsp90, in this study we decided to clone the gene for further application e.g., prevention of disease, diagnosis of infection and using as chaperonic molecule in other investigations.
MATERIALS AND METHODS
Parasite
Mice were infected via intraperitoneum by Toxoplasma tachyzoites
RH strain. Toxoplasma tachyzoites were isolated one week after infection
through mice peritoneum punction and rinsed by PBS buffer many times until ready
for RNA extraction.
RNA Extraction
Toxoplasma total RNA was extracted from tachyzoites under RNase free
condition described previously (Uckert et al., 1998) with brief modification.
Briefly, were mixed tachyzoites with 200 μL RNXplus buffer (CinnaGen
Iran) containing phenol and guanidine. The mixture was incubated for 5 min at
room temperature and then 50 μL of chloroform was added, mixed and centrifuged
at 12000 rpm for 15 min at 4°C. Supernatant (containing RNA) was transferred
to a new RNase free micro tube. Total RNA was precipitated by ethanol and finally
solved in 10 μL of diethyl pyrocarbonate treated water (Uckert et al.,
1998).
cDNA Synthesis
Reverse Transcription (RT) was performed under RNase free condition by using
5 μg of template RNA which was incubated in a 20 μL reaction mixture
containing 40 pico mol of Toxoplasma Hsp90 specific antisense primer
(HspR 5`-GAA TTC TTA GTC GAC CTC CTC CAT CT-3`), 100 unit of reverse transcriptase
enzyme (MMULV, Fermentas, Lithuania), 1x RT buffer, 20 unit RNasine (Fermentas
Lithuania) and 0.2 mM dNTP for 1 h at 42°C (Pfeffer, 1998).
PCR Reaction
PCR reaction was prepared in 50 μL volume containing 1 μg of synthesized
cDNA, 40 pico mol concentration of each forward and reverse Toxoplasma
Hsp90 specific primers (HspF 5`-GAG CTC ATA TGG CGG ACA CCG AGA CCT T- 3` and
HspR 5`-GAA TTC TTA GTC GAC CTC CTC CAT CT-3), 1.5 mM MgCl2, 0.1
mM dNTP, 1X PCR buffer, 1.5 unit of Taq DNA polymerase (CinnaGen, Iran). PCR
process was carried out with 30 cycles in thermocycler machine (Eppendrof, personal
model) under following conditions: denaturation at 94°C for 40 sec, annealing
at 65°C for 60 sec and extension at 72°C for 60 sec. Before PCR cycle's
initiation, reaction was incubated at 94°C for 5 min and after PCR cycle's
termination, reaction was incubated at 72°C for 5 min (Pherson and Moller,
2000).
Electrophoresis
PCR products were submitted to electrophoresis using 1% agarose gel. Gel
was stained by ethidium bromide and DNA band visualized under ultraviolet light
(UV transilluminator) (Boffey, 1984).
Gene Cloning
EcoRV blunt digested pBluescript and PCR product were electrophoresed in
1% Low Melting Point (LMP) agarose gel and expected DNA bands were sliced under
long wave UV. DNAs were recovered by DNA purification kit (Fermentas Cat. No.
k0513). Recovered PCR product and EcoRV blunt digested pBluescript were 3` tailed
using dATP and dTTP respectively by terminal deoxy nucleotidyl transferase (Eun,
1996; Gaastra and Klemm, 1984a). PCR product was ligated to pBluescript (Gaastra
and Hansen, 1984b) via T/A cloning method, transformed into E. coli XLI-blue
strain competent cells (Hanaham, 1983) and dispensed on LB agar plate containing
50 μg mL-1 ampicillin. Colonies were screened by X-gal and IPTG
to discriminate between recombinant (white) and no recombinant (blue) plasmids
(Bothwell et al., 1990). SacI and EcoRI restriction sites were established
on 5` end of forward and reverse primers respectively. Recombinant plasmid was
digested by SacI and EcoRI enzymes and a 2126 bp DNA band fragment encoded protein
of Hsp90 was released. Digested plasmid was electrophoresed in 1% low melting
point agarose gel and released DNA fragment (Hsp90 gene) was sliced under long
wave UV then recovered by DNA purification kit (Fermentas Cat. No. k0513). The
purified DNA fragment was subcloned in SacI and EcoRI digested PGEMEX-I expression
vector. The reaction was transformed in E. coli XL1- blue competent cells
and the positive colonies containing plasmid was mass cultured in LB medium.
Recombinant plasmid was extracted (Feliciello and Chinal, 1993) and confirmed
by restriction analysis.
RESULTS
Toxoplasma tachyzoites were isolated from peritoneum of infected mice and rinsed by PBS buffer. Total Toxoplasma RNA was extracted and cDNA was synthesized by specific Toxoplasma HSP90 antisense primer. PCR reaction was carried out by specific Toxoplasma sense and antisense primers.
The 2126 bp as expected PCR product of Toxoplasma tachyzoite Hsp90 gene is shown in Fig. 1.
PCR product was cloned in Bluescript plasmid and then subcloned in PGEMEX-I expression vector. Figure 2 shows recombinant compare with no recombinant plasmid.
Figure 3 shows electrophoresis of digested plasmid on LMP agarose gel. Gel was sliced and recovered by DNA purification kit, recovered DNA was 3` T tailed by terminal transferase and used for T/A cloning method.
PstI enzyme has one restriction site on HSP90 sequence but PGEMEX-I haven't restriction site for this enzyme. Therefore, recombinant plasmid was digested by PstI, but PGEMEX-I didn't. Figure 4 shows recombinant plasmid digested with PstI enzyme and confirmation of recombinant plasmid.
The PCR product of Toxoplasma gondii heat shock protein 90 was sequenced and deposited to GenBank under accession number.
| Fig. 1: | Electrophoresis on 1% agarose gel, Lane 1: 100 bp DNA ladder marker, Lane 2: PCR product of Toxoplasma HSP90 gene (2126 bp) |
| Fig. 2: | Electrophoresis on 0.8% agaroe gel, Lanes 1 and 3: No. recombinant plasmid, Lane 2: Recombinant plasmid |
| Fig. 3: | Electrophoresis on 1% LMP agaroe gel, Lane 1: 100 bp DNA ladder marker, Lanes 2 and 3: Digested plasmid |
| Fig. 4: | Electrophoresis on 0.8% agaroe gel, Lanes 1 and 3: Plasmid, digested with PstI (recombinant) Lane 2: Plasmid, undigested with PstI (nonrecombinant) |
DISCUSSION
Heat shock protein 90 (HSP90) excrete from extracellular tachyzoites of Toxoplasma gondii (Ahn et al., 2003). Although there are not enough informations about Toxoplasma gondii HSP90 molecule, but it must be important in toxoplasmosis. The 82 kDa heat shock protein (HSP90) is facilitating the entrance of tachyzoite into the host cells and its monoclonal antibody will protect this phenomena (Ahn et al., 2003).
Hsp90 is a chaperonic molecule which involves in different cell process like differentiation, apoptosis and cell proliferation (Heike et al., 1996; Sreedhar et al., 2004; Cox and Miller, 2003). This chaperonic molecule is very stable and has an important role in protein folding and some cell stresses (Zügelm and Kaufmannm, 1999). Heat shock proteins are used in vaccination in infection diseases (Zügelm and Kaymannm, 1999). It was used a DNA vaccine against Candida albicans in mouse (Raska et al., 2005) and also is a potential anticancer vaccine (Blachere et al., 1993). The Toxoplasma Hsp90 have cloned by Ahn et al. (2003) and they realized that Geldanamycin, a benzoquinone ansamycin antibiotic, known to interfere with HSP90 function, did not affect the secretion of Toxoplasma Hsp90 from extracellular tachyzoites. However, prevention of its expression will inhibit entrance of tachyzoite into the host cell as well as the intracellular proliferation (Ahn et al., 2003). Further studies are requisite about its function in living the parasite in host cell and its function as chaperonic molecule.
We like others (Ahn et al., 2003; Rojas et al., 2000) have cloned the heat shock protein gene of Toxoplasma tachyzoite named Hsp90 in expression vector and it is ready for further application e.g., prevention of disease, diagnosis of infection and using as chaperonic molecule in other investigations.
ACKNOWLEDGMENT
This study was supported by Vice Chancellor for Research of Shaheed Beheshti University of Medical Sciences and was done in Cellular and Molecular Biology Research Center. Here with, authors of research appreciating relevant responsible men.