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Information × Registration Number 0214U005542, 0113U000860 , R & D reports Title Obtaining of recombinant human collagen medications from plant systems in order to use them in ophthalmologic practice popup.stage_title Head Kuchuk Mykola Viktorovych, Registration Date 27-01-2014 Organization Institute of cell biology and genetic engineering NAS of Ukraine popup.description2 Structural parts of 1st and 2nd chains of I type human collagen and ? and ? subunits of prolyl-4-hydroxylase have been synthesized and cloned into vector constructs for bacterial expression (pQE-30, pQE-60, pET28a). The bank of plasmid DNA preparations and the corresponding lines of Esherihia coli XL1-Blue, BL21(DE3), C41, C43, Rosetta strains, that contain these vector constructs has been created. The synthesized genes were cloned into vector T-DNA for agrobacterial transformation. The bank of Agrobacterium tumefaciens GV3101 lines that contain vector constructs with collagen genes and prolyl-4-hydroxylase genes has been created. The transgenic Nicotiana benthamiana plants that contain genes of ? and ? subunits of prolyl-4-hydroxylase have been obtained via genetic transformation. The plant lines with both ? and ? prolyl-4-hydroxylase subunit genes have been selected by means of molecular genetic analysis. The transient expression of the genes of ?1 and ?2 collagen chains in N.benthamiana plants stably transformed by genes of ? and ? prolyl-4-hydroxylase subunit genes has been carried out. The protocol of N.benthamiana plant growning under the conditions of climate camera with strictly controlled parameters for simultaneous transient expression using the genetic constructions with ?1 and ?2 chains of I type human collagen and ? and ? subunits of prolyl-4-hydroxylase has been optimized. The protocol for detection and estimation of accumulation level of recombinant products in protein plant extracts by western-blot analysis has been worked out. The protocol for extraction and purification of the preparations of recombinant I type collagen by means of affinity chromatography methdod has been worked out. The obtained extracts of the purified preparations of recombinant protein have been analysed for the contamination presence, quantity parameter, have been characterized and transferred to the Filatov Institute of eye diseases and tissue therapy of NAMS of Ukraine for pre-clinical studies of the preparations. Product Description popup.authors Василенко Максим Юрійович Комарницький Ігор Климентійович Котик Богдана Євгенівна Лучаківська Юлія Сергіївна Петерсон Антон Андрійович Щербак Наталія Леонідівна popup.nrat_date 2020-04-02 Close