Cloning Of Porcine Growth Hormone Gene And Its Transfection In COS-7 Cells

In this research, the cDNA encoding porcine GH had been amplified by use of the total RNA,extracted from adult porcine's pituitary,as the template, Pl(5'-CTAGGATCCCCGGCTGTGATGGCCCTGCA)andP2(5'-CAGAATTCGCAA CAGAGATGCCCAG) as the primer pair and one particular cDNA product, 660bp, was obtained after the RT-PCR. The product was purified by using DNA agarose gel extraction method. After the purified cDNA and pMD18-T vector were ligated at 16℃ over night, the ligation products were transformed into E.coli strain DH5a and then the transformants were screened by clone PCR method. After sequencing of the positive clone and analyzed by BLAST on line, we found out that it encodes a deduced peptide of 216 amino acids(aa), with molecular weight 24.4KDa, isoelectric point at 7.26, and hydrophobic amino acids 44.9%, hydrophilic amino acids 30.6%, basic amino acids 13.0% and acidic amino acids 11.6%. It consists of 25aa making up the signal peptide and other 190 aa constituting the mature-peptide. In the latter, some residues differ from other reports on porcine GH, in detail, pGH in this study shares 100%, 99.1%,97.7% and 97.2% aa sequence similarity with the known sequences from other reports that their identification accession are respectively AAA31045 (Seeburg,P.H, et al,1983),CAA37411(Kato,Y,et al,1990), AAA73477 (Qi,S.-z,et al,1989), NP₉99034 (Chowdhary, et al ,1994), AAA73478(Qi,S.-z, et al, 1989).Porcine growth hormone (pGH) gene was correctly inserted into an eukaryoticexpression vector VR1020 in order to construct the expression plasmid pGH. The recombinant plasmid was transformed into E.coli DH5a competent cells, the positive clone was screened by Colony PCR and plasmid PCR and the recombinant plasmid was identified by BamH I and EcoRl digestion. Using lipofectin methods, the recombinant expression plasmid was transfected into COS-7 cells. RT-PCR> ELISA and Immunofluorescence assay was used to demonstrate that pGH gene has been be correctly transcripted and translated in COS-7 cells. This study sets a foundation for further research and application of recombinant GH in pig.