Всероссийский научно-исследовательский институт физиологии, биохимии и питания животных – филиал Федерального государственного бюджетного научного учреждения «Федеральный научный центр животноводства – ВИЖ имени академика Л.К. Эрнста»
ABSTRACT. After the creation of genetically engineered pharmacologic substances for human, a violation of tissue-specific expression of the transgene can occur, and getting these highly active substances in the blood of the fetus, which can lead to their death or to the emergence of various kinds of disease in offspring. The aim was to create a genetically engineered construction expressing in an inactive form a human granulocyte colony stimulating factor (hGCSF) in the mammary gland of mammals. The construction was created in which the structural human gene hGCSF is fused to the gene of human lactoferrin hLF. To create a new recombinant plasmid containing the gene hGCSF fused to hLF, the primers were constructed to hGCSF, containing a unique Bsu36I restriction site and a nucleotide sequence enabling the synthesis of amino acid sequences specifically hydrolyzable by bacterial protease (in forward primer). From the previously obtained plasmid pβLgGSSFcmvEGFP, DNA-fragment containing gene hGCSF was amplified and cloned into an intermediate vector pTZ57R/T. From transformedE. coli TG1 cells, the clones containing pTZ57R/GSSF were isolated. From pTZ57R/GSSF using a restriction enzyme Bsu36I (Eco81I), hGCSF gene fragment was allocated, providing formation amino acid sequences for bacterial IgA-protease. The plasmid was treated with restriction enzyme Pαs1Lf Bsu36I (Eco81I) and dephosphorylated with alkaline phosphatase. Fragment of GCSF/Bsu36I was ligated with plasmid pαs1Lf/Bsu36I/AlPh, then transforming cells E. coli TG1 were made. Grown on selective medium, the clones were verified by PCR-analysis for the presence of genes hGCSF and hLF. The selected clone was further subjected to restriction analysis. Plasmid pαs1Lf/GCSF is intended for production of transgenic animals that produce in milk the human regulatory proteins in an active form, which can be isolated after treatment by bacterial protease.
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