عنوان المقالة: Cloning of mutant staphylokinase by novel cloning vector M13 and PET32a from mutant Staphylococcus aureus and role in medical applications
Abstract
Study design of cases are Cross -sectional study design that descriptive study design and in analytical
case-control study design by internal comparison of 500 isolates collected from different provenance
of human infections including 280(56%) from tonsils , 100(20%) nose, 40(8%) tumors, 17(3.4%) urine,
27(5.4%) skin and 36(7.2%) blood, the isolation and identification achieved by using Vitek2-GP and
Genotypic detection of PCR to confirmative identification of S. aureus isolated from tumors.
Fibrinolytic enzyme (Staphylokinase) assay done on plasma agar plate and Casein agar plate to
determine fibrinolysis, caseinolysis on medium,
The first step of cloning occurs by using restriction enzyme BamH1 and EcoRI for cutting DNA M13
Novel cloning vector and plasmid vectors PET32a cloning vector is to grant sticky ends from DNA of
mutant S. aureus. The second step of cloning inclusive ligated by ligate digested fragment of DNA
mutant S. aureus (sak gene) with plasmid cloning vector with sticky ends that compatible with ends of
sak gene. The third step of cloning including transform recombinant vector (recombinant PET32a and
M13) into E. coli(DH5𝞪), the results showed positive transformants of E. coli(DH5𝞪) was selected on
plasma agar medium containing 50 mg/ml of ampicillin as a selectable marker for 20 isolates, the result
exhibit 14 isolates able to grown in presence of ampicillin and producing stapylokinase e nzyme.
Staphylokinase is recombinant drug therapy for hydrolyzing thrombosis. Purification of recombinant
staphylokinase from recombinant E. coli(DH5𝞪) by utilizing Ammonium sulfate, Dialysis, Gel
filtration and Ion Exchange chromatography of 14 transformants E. coli (DH5𝞪) express
staphylokinase from mutant S. aureus.
Study fibrinolysis achieved by using thrombolytic enzyme in vitro within tubes containing blood clot
(thrombi) to determine enzyme activity in vitro to hydrolyze thrombosis, results showed dissolve
thrombosis after adding 40,50,100μl from recombinant staphylokinase (thrombolytic enzyme) through
seconds.
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