Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Production Of Recombinant hGDNFIn HEK293T Cell Line SahbaShahbazi1, Liana Lachinani2, Kianoush Dormiani2, Yahya khazaie2, Kamran Ghaedi1,2,Mahboubeh foruzanfar2, Mohammad Hossein Nasr Esfahani2 1Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran 2Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran • Background and Objectives: • Glial cell line-derived neurotrophic factor (GDNF) which was first purified and characterized in 1993 is a disulfide linked homodimeric glycoprotein. This growth factor has neurotrophic effects on sympathetic neurons in the superior cervical ganglion and motor neurons in the spinal cord and protects midbrain dopaminergic neurons, and promotes their differentiation and survival, and It is synthesized in the form of pre-pro-GDNF which is proteolytically cleaved into a mature form thatconsist 134 amino acids. The aim of this study was construction of a bicistronic expression vector containing two copies of hGDNF for expression in a mammalian cell line. Method : Coding sequence of hGDNF was determined by bioinformatic studies. Two primer pairs were designed by means of Oligo7.4 software and two coding sequences of the hGDNF were amplified with the signal sequence at their N-terminus and 6xhistidine tag at their C-terminus. Using T/A cloning method, the fragments were ligated into pTZ57R/T vector. Each amplified hGDNF sequence with selected restriction enzymes subcloned into pBudCE4.1/attB, which is a 4.6 kb vector designed for simultaneous expression of two copies of genes in mammalian cell lines. The ligation mixture was transformed intoTOP10 E. coli competent cells. After isolation of positive colonies by colony PCR, the recombinant bicistronic vector was prepared and purified from transformed clones.ThenHEK293T cell line was stably transfected with Lipofectamine .The presence of the protein was confirmed by Western blot and SDS-PAGE followed by Coomassie blue staining. Results : The bicistronic expression vector was constructed successfully without any undesired mutation.ThenHEK293T cells were transfected and cell clones stably expressing hGDNF were isolated. The clones producing the highest amount of GDNF were expanded. Conclusions : The bicistronic expression vector was constructed successfully without any undesired mutation. By clonal selection, transfected lines that had higher expression rate of rhGDNF were isolated and after expanding the efficient clones, biological activity of secreted rhGDNF would be on going. Insert check PCR (A1 and A4 was selected as positive clones ) • References : • GarbayoAtienza E ,Blanco Prieto M., Aymerich M.S and Lanciego J. L .2006. Expression and Purification of GDNF for its microencapsulation in drug delivery systems and application in Parkinson’s disease . MAPFRE MEDICINA . 166-171. • 2. Garbayo E. , Ansorena E., Lanciego J.L. , Aymerich M.S. and Blanco-Prieto M.J. 2007. Purification of bioactive glycosylated recombinant glial cell line-derived neurotrophic factor . International Journal of Pharmaceutics 344 . 9–15. • 3.Ansorenaa Eduardo, Garbayoa Elisa, Lanciegob José L., Aymerichb Maria S., and Blanco-Prieto Maria J.2010. Production of highly pure human glycosylated GDNF in a mammalian cell line .International Journal of Pharmaceutics 385 .6–11. • 4. Chen ZY, He ZY, He C, Lu CL, Wu XF. 2000.A Structure-function Analysis of Human GDNF. sheng wuhuaxueyu sheng wuwu li xuebaoactabiochimica et biophysicasinica.243-247.