Cloning, Expression and Purification of Full-length Recombinant Ecarin and Comparing Its Expression and Function with Its Truncated Form.

Ecarin is a metalloproteinase found in snake venom (SVMP) with an important role in coagulation and control of hemostasis. It can specifically produce active-thrombin from prethrombin-2 and does not differentiate between normal and abnormal prothrombin. It is used in diagnostic tests and to evaluate the treatment process of many diseases. There are many drawbacks associated with separating these compounds from snake venom. Therefore, in this study, full-length recombinant Ecarin (r-Ecarin) was cloned, expressed, and purified in eukaryotic host cells. To determine the most effective form of the enzyme, r-Ecarin was compared with the recombinant truncated form, which has only the metalloprotease domain of the protein (r-Ecamet) in terms of function and expression. Briefly, A DNA construct composed of sequence-encoding Ecarin was designed and cloned into pCAGGS expression vector and, subsequently, expressed in Chinese Hamster Ovary (CHO) cells. To identify the enzymatic activity of expressed protein, a bioactivity assay was performed. Blood coagulation time and expression levels of r-Ecarin and r-Ecamet proteins were compared. Also, a histopathological assessment was carried out on the liver of mice treated with these proteins. Comparison of r-Ecarin and r-Ecamet expression pattern demonstrated that full-length Ecarin expression has at least 2-fold higher expression in eukaryotic cells. Determination of r-Ecarin function proved that this protein is capable of prothrombin cleavage and producing thrombin. Comparison of PT test results between the r-Ecarin and r-Ecamet showed that there is a significant difference in the activity of the two enzymes and the full-length protein coagulates the blood in less time.

Designing an Engineered Construct Gene Sensitive to Carbohydrate In-vitro and Candidate for Human Insulin Gene Therapy In-vivo.

Diabetes is a common disorder worldwide, and exhaustive efforts have been made to cure this disease. Gene therapy has been considered as a potential curative method that has had more stability in comparison with other pharmaceutical methods. However, the application of gene therapy as a definitive treatment demands further investigation. This study is aimed to prepare a suitable high- performance vector for gene therapy in diabetes mellitus. The designed vector has had prominent characteristics, such as directed replacement, which makes it a suitable method for treating or preventing other genetic disorders. The whole rDNA sequence of the human genome was scanned. The 800 bp two homology arms were digested by EcoRI, synthesized and cloned into the pGEM-B1 plasmid (prokaryotic moiety). The carbohydrate sensitive promoter, L-pyruvate kinase, and insulin gene were sub-cloned between homologous arms (eukaryotic moiety). The PGEM-B1 plasmid was digested by EcoRI, and the eukaryotic fragments were purified and transfected into Hela cell and then cultured. Afterward, the 300 µg/mL of glucose were added to the culture medium. Insulin expression in the transfected cells with 200 and 400 ng of the construct in comparison with negative control was detected using western blot and ELISA methods. Results have shown insulin expression in different glucose concentrates.