The production of the hormu00c8nio growth (GH) of Bos taurus by yeast Pichia pastoris using a synthetic gene sequence

摘要

The production of the hormu00c8nio growth (GH) of Bos taurus by yeast Pichia pastoris using a synthetic gene sequence. The object of the present invention is the production of recombinant bovine growth hormu00f3nio (rBGH) using the yeast P. pastoris expression system.The innovation is focused on the use of a synthetic gene sequence using preferential codons optimized GC nucleotide content and bases for P. pastoris. The production process of bGH recombinant expression system using the yeast P.Pastoris comprises: (a) the synthesis of cDNA of bovine GH encoder (Bos taurus taurus) using as a reference gene sequences described in GenBank (GI: 2168581, GI: 2168481, GI: 2168363) considering the following alterau00e7u00f3es: (1) the adaptation of sequ00fau00eancia synthesized of the hormu00f3n Io for the preferential codons of the yeast P.Pastoris and optimization of content of nucleotide bases go; (II) inclusion of restriction site xhol at the end 5 ', and Xbal, at the extremity 3', with the aim to ease the process of gene cloning vector of expression; (III) adding sequence of nucleotides that codifi Who to 6 amino acid residues at the 5 end immediately upstream of sequ00fau00eancia codante of bGH.And the addition of a stop codon downstream of the sequence; (b) cloning of the gene synthesized in vector expression / secretion in P. pastoris (pPICZ 244 ) by using methods of molecular biology; (c) processing the lineage of P.Pastoris gsii5 for extracellular expression of bGH; (d) cell culture of yeast in bottles of 1 litre for expression of the finned hormu00f3nio growth; (E) assessment of the expression of hormu00f3nio growth through Western blotting; (f) quantification of the efficiency of expression of Hor Recombinant human growth mu00f3nio per test of Bradford and densitometry of the gel.