عنوان المقالة: Cloning of mutant staphylokinase by novel cloning vector M13 and PET32a from mutant Staphylococcus aureus and role in medical applications
نبراس رضا محمد | Nebras Rada Mohammed | 7995
نوع النشر
مقال علمي
المؤلفون بالعربي
نبراس رضا محمد
المؤلفون بالإنجليزي
Nebras Rada Mohammed
الملخص الانجليزي
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.
تاريخ النشر
02/01/2021
الناشر
Nebras Rada Mohammed
رقم المجلد
24
رقم العدد
5
رابط DOI
DOI: http://doi.org/10.36295/ASRO.2021.24512
الصفحات
77-92
رابط الملف
تحميل (489 مرات التحميل)
الكلمات المفتاحية
Key words: Cloning, PET32a , Cloning vector, Recombinant drug therapy
رجوع