Suchergebnisse

Growth hormone (GH) is a polypeptide produced in the anterior pituitary lobe that triggers different biochemical pathways and increases cell proliferation and growth. In this work, the pNic based vector for periplasmatic secretion was developed. Recombinant human growth hormone was produced via periplasmatic secretion (42h, 30 °С) followed by several steps of purification. GH obtained does not contain His-tag. Expression level of 2,85 mg per1 literof bacterial culture was achieved.

A multiplex system to detect mutations at loci rs5985, rs1799983, rs5918, rs2243093, rs4673, rs4646994, rs1722009, rs3980933, rs71103505 associated with the development of cardiovascular diseases has been developed. These mutations belong to different types - SNP, STR, Ins/Del - therefore, minisequencing and fragment analysis technologies were used to determine them. Oligonucleotide analysis was performed to amplify all loci in a single reaction format. The minisequencing technology, in comparison with fragment analysis, required additional stages of sample preparation; therefore, oligonucleotides for loci with SNP were combined into a separate plex. For the two plexes created, the same composition of the amplification mixture and parameters of the PCR reaction program were optimized, and "bin" panels were developed to interpret the results. The testing of the system confirmed the possibility of detecting mutations at nine loci with high sensitivity and reproducibility.

A system has been developed to identify 11 genetic markers associated with the risk of obesity: rs10852521, rs11075990, rs1121980, rs1421085, rs1477196, rs17817449, rs3751812, rs7206790, rs8047395, rs9940128 (FTO gene) and rs1137101 (LEPR gene) by minisequencing (SNaPshot analysis). The conditions for carrying out the amplification and minisequencing reactions, as well as the compositions of the reaction mixtures, were optimized so that the analysis was carried out for all 11 markers simultaneously. The resulting system was tested and showed a high degree of reproducibility and sensitivity required for the detection of these polymorphisms.