Rekombinant yöntemlerle sentetik parathormon üretimi

Abstract

Proteins have a great importance within the field of biotechnologically derived products. Synthetically produced proteins using recombinant methods are produced for therapeutic, industrial and scientific research purposes. The aim of our work is to produce parathormone, which we aim to use therapeutically using recombinant technology. Parathormone (PTH) is secreted by the parathyroid glands as a single chain of 84 amino acids (aa) in the polypeptide structure. The primary responsibility of PTH is to regulate plasma calcium (Ca+2) levels by Ca+2 absorption from the bones. It has an important role in providing homeostasis in Ca+2-PO4−3 metabolism by providing kidney and intestinal Ca+2 absorption as well as phosphate (PO4−3) excretion. Parathyroid hormone (PTH) promotes absorption of calcium from the bone in 2 ways. The rapid phase brings about a rise in serum calcium within minutes and appears to occur at the level of the osteoblasts and osteocytes and when PTH binds to receptors on these cells, the osteocytic membrane pumps calcium ions from the bone fluid into the extracellular fluid. The effect of vitamin D on the production of the active form facilitates absorption of calcium from the intestines. More or less secretion of the hormone can cause a variety of health problems. The most important endocrine discomfort associated with low secretion of PTH is called hypoparathyroidism. In our study, planned synthetic PTH production was targeted as an alternative treatment method of hypoparathyroidism. In this study, the PTH gene was obtained as a result of isolation of messenger ribonucleic acid (mRNA) from the sample taken from the hyperplasia tissue and subsequent polymerase chain reaction (PCR) experiments using DNA polymerase enzyme, which generates DNA fragments with specific primers of complementary deoxyribose nucleic acid (cDNA). The gene encoding the recombinant PTH (rPTH) N-Terminal (1-84) was placed behind the secretion signal in the cloning vector of PEGFP-N1 (eukaryotic plasmid vector) and cloned using molecular cloning techniques. As a host organism One Shot® Mach1™ T1R Escherichia coli competent strain was used. The rPTH gene introduced into the vector is introduced into the cell culture with HeLa (Henrietta Lacks, cervical cancer) and transfection agent on AGS (ATCC® CRL-1739 ™, Gastric Adenocarcinoma) cell lines for 24 h, 37 celsius (˚C) left to incubate. After 24 hours, intact parathormone level in the culture fluid was measured with activation measurement and expression level was evaluated with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results acquired in this study and ongoing synthetic parathormone development studies in the medical scope for N-Terminal (1-84) PTH formulation, it was aimed to promise for hypothyroidism treatment and to contribute to scientific researches in the recombinant medicine production.