Alan, Jaime Jacqueline Ja Yap
(2005)
Cloning and mutation of chinese hamster
ovary cell elongation factor (cej) of
vibrio cholerae.
Masters thesis, Universiti Sains Malaysia.
Abstract
Cholera caused by toxigenic Vibrio cholerae is a major public health problem
confronting developing countries, where outbreaks occur in a regular seasonal pattern
and are particularly associated with poverty and poor sanitation. The disease is
characterized by a devastating watery diarrhea which leads to rapid dehydration, and
death occurs in 50 to 70% of untreated patients. Prophylactic vaccination for cholera
was initially thought as a means of control programme. Although cholera vaccine
strains have been constructed with all known toxin (El Tor hemolysin, CT, Zot, and
Ace) genes deleted or inactivated, some volunteers fed with these strain still develop
diarrhea probably due to the presence of cell- associated CHO cell- elongating factor,
cef
In this study we therefore, have tried to clone and mutate this cef gene with the
hope of eliminating its residual diarrheagenic effect, thus the development of improved
VCUSM2 and VCUSM4 vaccine candidates. Accordingly, the cef factor was amplified
from wild type Vibrio cholerae which was subsequently cloned into a cloning vector,
pTZ57R/T at Eco321 site. The cef gene in pTZ57R was digested with Psyl
enzyme, to allowing insertional mutational ligation to occur at this site. The PCR
amplified kan resistance gene cassette from pTOP02.1 was used to insertionally
mutate the cef gene as well as a marker for selection fallowing transformation. The
selected clones with kan marker were screened by PCR for confirmation and
subsequently verified by DNA sequencing
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