Production of recombinant urease for screening of helicobacter pylori infection
Helicobacter pylori establishes infection inside human stomach lining and causing duodenal diseases, such as peptic ulcer and potentially into gastric cancer. The invasive diagnostic methods require unpleasant endoscopic procedure for H. pylori detection. Preferably, the noninvasive diagnostic me...
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| Format: | Thesis |
| Language: | English |
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| Online Access: | http://dspace.unimap.edu.my:80/xmlui/bitstream/123456789/44263/1/p.1-24.pdf http://dspace.unimap.edu.my:80/xmlui/bitstream/123456789/44263/2/full%20text.pdf |
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| Summary: | Helicobacter pylori establishes infection inside human stomach lining and causing
duodenal diseases, such as peptic ulcer and potentially into gastric cancer. The
invasive diagnostic methods require unpleasant endoscopic procedure for H. pylori
detection. Preferably, the noninvasive diagnostic methods would make suspected
patients less stressful to procedure for H. pylori detection. The two most preferable
noninvasive diagnostic methods are antibody detection (serology) and antigen
detection (from fecal) with their own advantages and drawbacks. H. pylori urease is
one of the antigens found in H. pylori with strong immunogenic property. Thus,
urease was chosen for the development of H. pylori dot-EIA test strip which could be
used in a serology based detection system for H. pylori infection. Cloning of urease
A gene fragment (pET32ureA3), urease B gene fragment (pET32ureB2) and the
whole of urease operon (pET32UOA6) produced biologically active recombinant
urease A (UreA), recombinant urease B (UreB) and recombinant urease enzyme
complex (UreA/UreB) verified by immune functioning assay using commercial
antibody H. pylori urease-α and commercial antibody H. pylori urease-β from Santa
Cruz, Inc, USA. The production of recombinant UreA/UreB complex indicates that
a fully functional urease operon or a urease replicon was successfully constructed.
Purifications of the recombinant ureases were successful and the purified
recombinant ureases were still biologically active. The purified recombinant ureases
were coated onto membranes in preparation of dot-EIA test strips. The prepared dot-
EIA test strips gave brown colour dots indicating positive reactions when probed with commercial antibodies against ureases (Santa Cruz, Inc, USA). Nine rabbits
were used to assess the immunogenicity properties of the recombinant ureases. Sera
from the challenged animals gave positive detections on the prepared test strips,
similar to detections using commercial antibodies, indicating the recombinant
ureases could act as immunogens comparable to native ureases. Three groups
containing three rabbits each were challenged with purified recombinant UreA, UreB
or UreA/UreB respectively showed the presence of ureases antibodies in their sera on
dot-EIA test strips. One rabbit that served as a negative control, challenged with
bovine serum albumin (BSA), did not give positive reaction on dot-EIA test strip.
To conclude, this study was successfully achieving all the objectives: cloning,
expression and purification of functioning recombinant ureases, as well as,
developing dot-EIA test strip. With the intention to develop user friendly, easy,
cheap and fast detection; this dot-EIA test strip provides a foundation for further
urease enzyme-linked serology based assay development as a mean for early
screening. In addition, the constructed H. pylori urease replicon opened an
opportunity for developing a genetically modified animal model to study H. pylori
pathogenesis. |
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