Molecular cloning, sequence analysis and eukaryotic expression of sheep growth hormone gene.

Total RNA of pituitary was extracted from Xinjiang Altay×Chinese Merino F1 sheep.The cDNA encoding growth hormone(GH) gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR) method using isolated total RNA as template.The product of RT-PCR was inserted to plasmid pT-Adv by T-A cloning.The result of nucleotide sequencing showed that the cloned cDNA had positive reading frame and its length was 654 bp.Its nucleotide sequences were compared with three piece of published ovine growth hormone gene(oGH) cDNA sequences and the result showed that there were differences between 7 bases.These variation are all silent except the two base changes found at 472 and 529 nt.This suggested that the oGH gene from Altay × Chinese merino cotains polymorphisms between different breeds.The oGH cDNA was subcloned into the eukaryote expression vector plasmid pcDNA3.1A and constructed the recombinant eukaryote expression plasmid pcDNA3.1A/oGH cDNA.By liposome transfection,the recombinant plasmid were transferred into SP2/0 cells.The result of indirect ELLSA suggested that there were recombinant oGH protein in the cultural supernate of the transfect cells.