Ten sub-fragments (N-, P-, C-, NS-, NP-, NPC-, S-, SP-, SPC- and PC-domains) of recombinant S. mansoni calreticulin (SM-CRT), a highly immunogenic antigen, were expressed and purified. A cDNA library of adult S. mansoni was used as a template to amplify the coding sequences of these fragments which were expressed in bacterial expression system using the vector pRSETB. SM-CRT sub-fragments (except those with N-terminal domain) were shown to bind calcium using “Stains-all” stain or interact with 45Ca2. With the exception of the P-domain, SM-CRT sub-fragments bound and inhibited C1q-dependent haemolysis in vitro. In S. mansoni infected mice, specific antibodies against the carboxy-terminal part of SM-CRT appeared 46 days p.i., while antibodies against the N-terminal part of SM-CRT were detectable at 59 days p.i.. Cercarial Transformation Fluid (CTF) and Solube Egg Antigens (SEA) specific antibodies were detectable at day 12 p.i.. Use for diagnosis of S. mansoni in humans, CTF and SEA showed 89.7% sensitivity, while the PC-domain of SM-CRT showed sensitivity of 71.1% (the highest SM-CRT sub-fragment sensitivity). When testing the cross-reactivity with sera drawn from patients with other diseases, SM-CRT N-domain showed the lowest degree of cross-reactivity. Analysing non-endemic controls, a specificity >95% was shown for all of the antigens used. When using endemic controls, SM-CRT N-domain revealed the highest degree of specificity (94.7%), while SEA revealed 26.3% specificity and CTF showed 68.4% specificity. BALB/c mice immunised with recombinant SM-CRT achieved a 49.9% (P >0.05) reduction in Schistosoma adult worm numbers (on exposure to S. mansoni cercariae), comparing to the non-immunised mice. The eggs numbers in the liver decreased by 41.8% in the immunised mouse group with a non-significant statistical difference. The immunised mice responded to SM-CRT immunisation by producing specific IgG1 and IgG2a, reflecting a Th1/Th2 with a predominance of Th2 immune response profile.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:551899 |
| Date | January 2010 |
| Creators | ElAswad, Bahaa El-Deen Wadea Ramadan |
| Contributors | Schwaeble, Wilhelm |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/9961 |
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