Genomic Investigation and Cloning of the Fibrinolytic Gene (bsfA) of Thermophilic Soil Bacillus in Escherichia Coli Origami by Molecular Method, Real Time PCR and SDS Technique

Background and aim: Enzymes play a very important role in various industries and in medicine. The BSFA genomic region is also considered one of the most important operons in biotechnology and bioengineering and is used in the preparation of fibrin-breaking drugs (fibrinolytic) or clot-breaking drugs to destroy blood clots in vessels (thrombosis and heart attack treatment) and this enzyme can be used in therapeutic cases. The thermophilic bacillus carries various enzyme genes and the goal is to investigate the genomics and cloning of the fibrinolytic gene (bsfA) from soil thermophilic bacilli in Escherichia coli origami bacteria by Real time PCR and SDS PAGE methods. Materials and Methods: Bacillus strains were isolated and identified from a total of ۷۰ soil samples from the areas around Tehran. Then bsfA gene was extracted from bacilli by PCR method. The amplified fragment was inserted into the pTG۱۹ expression vector by TA cloning (Transfer activity) method. In the next step, the recombinant vector was transformed into Escherichia coli origami bacteria and cloning was confirmed using common methods. PCR reaction was performed for the bsfA gene with the mentioned primers. Results: As a result, all of the ۱۲ Bacillus strains isolated (۱۰۰%) had the bsfA gene. The results of RNA cloning of suspected colonies extracted cDNA was made and the expression level of bsfA gene was checked by real time PCR test. Determining the molecular identity of the Bacillus genus carrying the bsfA gene was done using primers. Finally, the expression of the bsfA gene in Escherichia coli origami bacteria was confirmed by the sequence of the PCR product. Conclusion: As a result of this research, they managed to find native bacilli producing bsfA, and finally, successfully transferred the gene of this enzyme from Bacillus bacteria to E.coli bacteria so that this enzyme can be produced in E.coli with more production and economic efficiency. To create bacteria with high expression and recombination ability, it can be a big step in increasing the production of this enzyme in the treatment of patients suffering from thrombosis, which prevents embolism and death.

کلیدواژه ها:

bsfA gene ، Bacillus ، cloning ، Fibrinolytic ، Real time PCR method ، SDS-PAGE technique ، ژن bsfA ، باسیلوس ، کلونینگ ، فیبرینولیتیک ، روش Real time PCR ، تکنیک SDS-PAGE