Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris

Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.

Restriction enzyme digestion analysis of PCR-amplified DNA of Blastocystis hominis isolates.

Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.

Acute cholecystitis caused by Clonorchis sinensis.

We are reporting a case of a 43-year-old Chinese male from Hong Kong, who came to see a doctor complaining of acute onset of severe upper abdominal pain. A diagnosis of acute cholecystitis was made and an emergency cholecystectomy was carried out. On opening the common bile duct, lancet-shaped worms were seen emerging from it. About 45 adult worms were collected and sent to the Department of Parasitology University of Malaya for identification. The worms were identified as Clonorchis sinensis. After the operation the patient was treated with praziquantel and he had an uneventful recovery.

Polypeptides associated with in vitro cyst formation of Blastocystis hominis.

The objective of this study was to characterize the polypeptides associated with cysts of Blastocystis hominis. This form is believed to be infective and plays a role in parasite resistance to anti-B. hominis drugs currently used for treatment of Blastocystis associated diarrhea. Cysts were induced through in vitro culture of the parasite in complete medium supplemented with bacterial extract with trypticase, metronidazole or doxycycline. SDS-PAGE analysis showed almost similar polypeptide patterns of parasite extracts obtained from in vitro cultured parasites before and after exposure with the three supplements. Polypeptide bands at 76, 58.5, 48, 45, 40, 38, 32, 25 and 22 kDa were constantly seen in all antigenic preparations and no specific cyst-associated polypeptide was present. However, on immunoblot analysis, 3 out of 16 blastocystosis human sera identified a cyst-associated polypeptide at 60 kDa in all parasite extracts prepared from cultures with the three supplements. In addition, there were associated morphological changes detected in these parasites stained with acridine orange and observed under fluorescence microscopy. Metronidazole induced cyst forms (reddish cells) as early as 12 hours post-exposure; more cyst production (with stronger immunoblot bands) occurred after 24 hours exposure. However, cysts rupture with release and destruction of B. hominis daughters cells occurred after 48 hours exposure. Doxycycline induced less cyst-like forms at 24 hours (weaker 60 kDa band) and less destruction of the cysts (60 kDa band still present at 72 hours post exposure). Bacterial extract and trypticase also induced cysts at 12 hours with increasing numbers up to 72 hours exposure (corresponding increase in intensity of 60 kDa band from samples harvested at 12 to 72 hours post exposure) without any sign of deleterious effect on the parasite.