A process for obtaining a kunitz protease inhibitor from an amblyomma cajennense tick salivary gland cdna library: clone oligonucleotide sequence, and recombinant protein amino acid sequence, recombinant protein; process for determination of inhibitory activity on activated factor x, process for determination of anticoagulant activity in plasma, process for determination of apoptotic activity in human and murine tumor cell lines, process for determination of anti-metastatic activity in melanoma tumor, process of in vitro and in vivo anti-cancer activity determination, recombinant use in different homeostatic (coagulation and immune response), anti-proliferative, anti-apoptotic and anti-angiogenic mechanisms

摘要

"PROCEDURE FOR OBTAINING A KUNITZ TYPE PROTEASE INHIBITOR FROM A CDNA LIBRARY OF THE AMBLYOMMA CAJENNENSE CARNATE: RECEIVING PROTEIN CLEANING AND SEQUINED PROQUINES ON THE ACTIVATED FACTOR, PROCESS FOR DETERMINING ANTICOAGULANT ACTIVITY IN PLASMA, PROCESS FOR DETERMINING APOPTOTIC ACTIVITY IN HUMAN AND MURIN TUMOR CELL LINES, PROCESS FOR DETERMINATING ANTI-ACTIVITY DETERMINATION CANCER, IN VITRO AND IN VIVO, USE OF RECOMBINANT IN DIFFERENT HOMEOSTASTIC MECHANISMS (COAGULATION AND IMMUNOLOGICAL RESPONSE), ANTI-PROLIFERATIVES, ANTI-APOPTOTICS AND ANTI-ANGIOGENS ". The present invention is a recombinant Kunitz-type inhibitor that was obtained from a cloned gene from an Amblyomma cajennense salivary gland cDNA library, and the inhibitor named Amblyomin-X has a molecular mass in the order of 13,500 Da. Amblyomin-X when tested on FXa using FXa specific colorimetric substrate inhibits FXa and inhibition was dependent on the presence of phospholipids (Phosphatidylserine: Phosphatidylcholine). Amblyomin-X inhibitory activity was also verified in the coagulation tests, and the activated partial thromboplastin time (TTPA), the prothrombin time (TP) and the PCA - pro coagulant activity assay were evaluated. The presence of 7.5 µM inhibitor prolongs about 3-fold TTPA; approximately 6-fold TP, and also extends about 2-fold PCA, and this assay was performed both in the presence of phosphatidylserine: phosphatidylcholine and in the presence of apoptotic bodies (made from CHO cells) as a source of phospholipids. In vitro treatment of the B16F10, SW12A1, B3, L292, MCF7, HL60, K562, U937 and JURKAT tumor lines with Amblyomin-X has shown that the inhibitor exerts a time and dose dependent apoptotic effect on these cells. No effects were observed on normal human fibroblast cells, macrophages, neutrophils and lymphocytes. Mice (C57BL / 6J) with dorsal melanomas receiving Amblyomin-X (1mg / kg) for 12 days showed significant reduction in tumor mass and number of metastases when compared to a group of untreated tumor bearing animals. In addition, mice (C57BL / 6J) with lung metastases treated with Amblyomin-X (1mg / kg) for 12 days showed a reduction in the number of tumor nodules in the lung parenchyma when compared to an untreated tumor group. Subcutaneous treatment of melanoma-bearing mice (C57BL / 6J) with 42 doses of Amblyomin-X (1mg / kg) showed that the inhibitor is capable of complete tumor remission, prevent metastasis and not cause hematological alterations in the animals, keeping the blood count similar to that of a group of healthy animals. Cell cycle analysis of the dorsal tumors of Amblyomin-X treated animals showed a large proportion of quiescent, low proliferative apoptotic tumor cells. The cells obtained from pulmonary metastases are largely in the G2 / M phase unable to divide. The functional activity of macrophages obtained from dorsal tumor animals treated with Amblyomin-X showed that the drug increases the phagocytic capacity of these macrophages mediated by C3B and FC receptor. Thus, Amblyomin-X, besides being an FXa inhibitor, is also effective in the targeted treatment of malignant neoplasms.