Lutjanus Erythopterus GH And IGFⅠ Gene Clone And Eukaryotic Expression

Growth Hormone (GH) is a peptide produced by vertebrated animals’ hypophysis;growth hormone of fish can promote fish growth and their metabolism. Insulin-like growthfactor Ⅰ(IGFⅠ) is one kind of multifunctional cell proliferation regulation factor,produced by pancreas, it is very important in cell growth and development of fish.Naturally，The two factors in fishes’ bodies is extremely low, it is difficult to purify them,gene engineering technology can save the problem, to produce a lot of cheaper fishesgrowth hormone. Restructuring fish growth hormone has the same function of naturalgrowth hormone. In aquaculture, lots of fishes’ growth hormone have been used, and hadMade Profit. Therefore, it’s valuable to poduce restructuring growth hormone of fish. Wesuccessfully gained crimson snapper’s growth hormone gene and insulin-like growth factorgene, and eukaryotic expression crimson snapper growth hormone (Lutjanus erythopterusGH) in Pichia pastorios, and feed the tilapia with Restructure Pichia pastorios (whichcontains lGH protien), primary result shows the recombinant growth hormone can promotetilapia growth.(1) Clone GH gene and IGFⅠ gene of crimson snapperDownload GH and IGFⅠ gene sequence similar to crimson snapper in Gene Bank,designed appropriate primer in conservative district of the two genes. PCR amplificationobtain partial coding areas sequence of GH and IGFⅠ,GH467bp and IGFⅠ506bp.Used specific primer joint extending their3’ and5’ end, as a template to PCRamplification, then cloned them to plasmid, then sended to Shanghai Sagon Companydirect sequencing. At last, we spliced them by Congtig Express, and analysed theirsequences online. Their sequences features as follow: GH Gene is787bp, it’s5’UTR19bp, coding-region615bp,3’UTR143bp, submitted to the Gene Bank, registration numberof is: JQ346228; IGF Gene is1132bp, it’s5’UTR134bp, coding-region558bp,3’UTR460bp, submitted to the Gene Bank, registration number of is: JN383435.(2) GH of crimson snapper restructuring in pichia pastorios and its promoting growtheffectCloned crimson snapper’s GH coding-areas cDNA to the expression vector(pPICZαA), constructed the restructuring expression plasmid pPICZα-GH. Transformed the plasmid to Pichia pastorios GS115, with the YPDZ medium screened and PCRamplified the restructuring Pichia pastorios, induced they expression, SDS-PAGE showedthat the GH protein expressed. The better codition for GH expression is: inducing at30℃,methanol0.5%, pH at6.0. Used restructuring Pichia pastorios feeded tilapias,25daysprimary test shows that mixed culture of restructuring GH Pichia pastorios can promotetilapias’ growth.