Female was linked to T cell epitopes with

Female
6 to 8 week old BALB/C mice were purchased from Pasteur Institute of Iran. All
experimental procedures were conducted according to animal guidelines ethics.
The study was approved by the Ethics Committe of Iran university of medical
sciences. (Approval No..9221577201  )2.2.
Toxoplasma gondii strainsThe
high virulence T.gondii RH strain was provided by the toxoplasmosis
research center from Tehran university of medical sciences, Iran. BALB/c mice
were injected via peritoneal cavity with 1×105 of  tachyzoites suspended in sterile
Phosphate-Buffered Saline (PBS; pH=7.4) comprising 100 mg/ml streptomycin and 100
IU/ml penicillin. Tachyzoites were harvested after  3-4 day from peritoneal cavity of mice. (GRA14).2.3.
Preparation of Toxoplasma Lysate Antigen (TLA)The
toxoplasma lysate antigen (TLA) was set up as described previously (ROP18 Rop5). Briefly, Peritoneal
cells were washed with PBS and centrifuging at 800×g for10 min at 4°C. the
purified tachyzoites were disrupted by ten cycles of freezing and thawing in
liquid nitrogen. Lysate was centrifuged at 10000g for 20 min at 4°C then
filtered with 0.22 ?m sterile filter. The total protein was assessed by BCA (bicinchoninic
acid) method. TLA specimen were aliquoeted and stored at -80°C.2.4. Bioinformatic implements for
predicting T-cell epitopes T cell epitopes of ROP2, GRA4 and AMA1
of T. gondii RH strain were predicted with bioinformatics softwares of
IEDB. Among the predicted epitopes, the 8 T-cell epitopes and 2 MHC II  fragments were chosen due a higher score of
mentioned genes related to a higher possibility of the residue being involved
in the epitope, using Database online service
IEDB(http://tools.immuneepitope.org/mhcii) (Table 1).2.5. predicting B-cell binding epitopeBcell epitope of SAG1
were chosen in previous study by A.Stuara (). The epitope detectd at the surface of
the D1 domain from monomeric form of SAG1 molecule that compound to a Fab
monoclonal antibody. This antibody 
increase against tachyzoite strongly. So, this epitope is an immunogold
region on the surface of SAG1.Multi-epitopes gene synthesis                                                                                                         
B
cell epitope of SAG1 was linked to T cell epitopes with GGGGS  linker encoding amino acids. Consequently the
Toxoplasma gondii multi-epitopes gene (TgMEG) was engendered using
chemical gene synthesis. TgMEG also comprised an enzyme restriction Nde1 sequence, AAT start codon, following a
TAA stop codon and an enzyme restriction Xho1 sequence and with double six histidine residues tag
(6×His). TgMEG
, a total of 700 bp, was
synthesized and cloned in the  pET28a
vector by Pepmic Co, Suzhou China.2.5.
Expression and Purification of recombinant protein

The
E. coli strain of BL21 (DE3) (Novagen, Darmstadt, Germany) cells were
transformed with pET28b/MEG and were grown in LB broth medium with 100mg/ml
kanamycin to an optical density of 0.6 at 600nm. Enhance the solubility of
target protein with  0.5 mM of IPTG (Isopropyl
?-D-1-thiogalactopyranoside) (Sinaclon, Iran) at 37°C, followed by harvest of
the bacterial cell by centrifugation at 4000 rpm for 30 at 4°C and lysis. The
expression products were disrupted by freeze-thawing and centrifugation at 13,000
g, 4°C for 20 min. proteins were produced in insoluble forms accumulated in
inclusion bodies, as estimated by 12% SDS-PAGE electrophoresis, they were
isolated under denaturing conditions with 6M urea. The solubilized proteins were purified by affinity
Ni2+-chromatography (Qiagen, Hamburg, Germany), according to the manufacturer’s
instructions. After purification the recombinant proteins were refolded, analyzed
by SDS-PAGE electrophoresis. Protein Ladder (Sinaclon, Iran) were used as a molecular
weight marker for SDS-PAGE. Finally, the purified protein concentration was
assessed by a BCA protein assay reagent.

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