Asian Research Thesis Index

Abstract

Tuberculosis (TB) is said to be the oldest disease of history and still considered as a
major problem in the world especially in developing countries like Pakistan. It needs
continuous efforts to work on its prevention and cure. Mycobacterium tuberculosis (M.
tb) bacterium is the etiological agent of this disease and almost one third population of
world is infected with it. Almost 1.5 million deaths/year are due to active TB. Asia
carries two-third of the total burden of TB, and Pakistan is at 5th position among 22
high burden countries. Efforts are made to control and prevent TB by improving
diagnostic tests, effective medication, care through DOTS program and development of
new vaccines. BCG vaccine is available as control measure but its efficacy is very
limited in most parts of the world. There is need to develop better and state of the art
vaccines like DNA vaccines. The current study was designed to produce construct of M.
tb genes and later used them as DNA vaccine in mice model. The two M. tb genes SodC
and Mpt51 were selected and their gene sequence was downloaded from Tuberculist
database. Primers were designed manually. PCR conditions were optimized for each
gene and PCR products were confirmed through restriction digestion. PCR products
were cloned into pTZ57R/A vector and transformed into chemically competent E.coli
cells. The clones were confirmed through restriction digestion and finally by sequence
analysis. Finally the constructs were made in pND vector. The finally confirmed clones
were stored at -70
C as glycerol stocks and later were used for sub-cloning purposes in
mammalian expression vectors to finally develop DNA vaccines. In this regard, twenty
Balb/c female mice were used having age of eight weeks each. Eight eight animals were
used for inoculation with SodC-pND and Mpt51-pND respectively. Four were used as
negative control (Normal Saline group). All animals were bled through tail after three
weeks intervals and finally through cardiac puncture at nine weeks post inoculation. All
antisera were strongly positive on dot blot and Agar Gel Immunodiffusion test. The
results of this study proved that both SodC-pND and Mpt51-pND DNA vaccines are
good enough to produce strong humoral response in mice model and hold a promise to
be used in anti-TB vaccines in future

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