Genetic Engineering of the Cloning Mutant Müller's Factor Gene Using Physical Mutagens of Alpha, Beta, Gamma Rays Emitted from CS137, CS138, Am241, Sr90, Sr91 and UV. Radiation Via Puc and M13 Cloning Vector Within Competent Bacterial Strain NM522 to Prod

Abstract

Objective: The aim of the research is to cloning the Müller's factor gene after exposing it to various physical mutagenic agents from radioactive sources and ultraviolet radiation to increase production of thrombolytic enzyme with application in medical therapy such as thrombosis.
Backgrounds: S. aureus gram positive bacteria, produces many virulence factors such as hemolysins, leukocidins, proteases, enterotoxins, exfoliative toxins, fibrinolytic enzyme and immune-modulatory factors. Thrombolytic enzyme is one of stronger factors released from S. aureus strains, expressed by lysogenic strains that degradation fibrin clot. It is known as thrombolytic enzyme, proteolytic enzyme, Staphylococcal fibrinolysin and Muller's factor.
Methodology: One hundred Staphylococcus aureus isolates were obtained from blood, tumors, cancer, pus, skin lesions and other sources. These isolates were exposed to various radioactive sources including Cs137, Cs138, Sr90, Sr91 and Am241 at different doses and for varying durations including alpha, beta and gamma radiation, also exposure to UV light at different time. The number of viable cells, the percentage of cell death, antibiotic activity and the production of thrombolytic enzymes were calculated before and after exposure. Polymerase chain reaction (PCR) was performed on the sak gene extracted from the mutant S. aureus. The PCR product was genetically engineered and the enzyme was extracted and purified from the bacteria in which the genetic engineering was successful. The lysis of blood clots was examined in vitro and also in mice.
Results and Discussions: The results of the antibiotic and fibrinolysis enzyme tests for the mutant S. aureus after exposure to alpha, beta, gamma and ultraviolet radiation showed that some of the bacteria that remained alive lost their resistance to antibiotics and became very sensitive. The fibrinolysis enzyme production test showed that its production became many times greater than before exposure. The sak gene was extracted from all the mutant S. aureus and the product of the polymerase chain reaction was cloned into a genetically engineered bacterium NM522 using the M13 cloning vector and the pUC plasmid cloning vector. The results of the genetic engineering showed that 8 isolates of the genetically engineered NM522 bacteria had white colonies which is proof of the success of the cloning process. The enzyme was extracted and purified from genetically engineered bacteria. The clot formed in the laboratory was examined for human blood and the clot was examined inside mice. The results showed that the blood was restored to its liquid state and the clot was dissolved quickly within seconds. Likewise, inside mice, the blood was dissolved within a very short period after the process of inducing the formation of the clot was carried out inside the mice and examined after injecting the enzyme inside the mice.
Conclusions: The mutant bacteria transitioned from antibiotic-resistant to antibiotic-sensitive. The mutant bacteria transitioned from low or non-producing to producing a high level of the thrombolytic enzyme. The cloning process in genetic engineering successfully engineered the mutant gene from the mutant bacteria, resulting in the production of a highly effective thrombolytic enzyme for thrombosis and the treatment of blood clots and strokes.