Process for obtaining high nu00ecveis expression of hormu00c8nio stimulator of thyroid (tireotrofina human or hTSH) in Chinese hamster ovary cells.By the use of vectors for expression dicistru00c8nicos,Application of an amplification strategy with double marker gene detection and quality control of the same hTSH throughout the production process by high performance chromatography lu00ecquidaReverse phase

摘要

"The process for obtaining high nu00ecveis expression of hormu00c8nio stimulator of thyroid (tireotrofina human or hTSH) in Chinese hamster ovary cells.By the use of vectors for expression dicistru00c8nicos,Application of an amplification strategy with double marker gene detection and quality control of the same hTSH throughout the production process by high performance chromatography lu00ecquidaReverse phase ".The process uses the cells of Chinese hamster ovary (CHO),It is based on the use of vectors for the expression dicistru00f3nicos subunits 244 and 225 of the hTSH and markers of gene amplificu00e1veis diidrofolato reductase (DHFR) and adenosine deaminase(ADA). Were isolated the genes coding for the subunits of and 225 244 this heterodimeric glycoprotein and constructed their expression vectors (peddc - 244 and hTSH peadc 225).Next, cells deficient in CHO DHFR (dbfr ^ - ^) were cotransfectadas with these vectors and cultured in selective medium.The isolated clones were subjected to sequential gene amplification in medium containing increasing concentrations of methotrexate (MTX) and deoxicoformicina (DCF).With the use of MTX were obtained levels of secretion of 7.2 sym 1.3 109 G hTSH / 10 ^ 6 ^ cells / day.These levels were further increased after the use of the marker ADA upon treatment with DCF, reaching levels of 17.8 sym 7.6 109 G hTSH / 10 ^ 6 ^ cells / day.Therefore, to obtain high protein yield was mainly due to the original strategy of amplification using double marker gene.