Study On Cloning And Prokaryotic Expressing Of Growth Hormone CDNA Of Ctenspharynogodom Idellus

Total RNA was isolated from grass carp (Ctenspharynogodom idellus) pituitary. The cDNA encoding growth hormone (GH) peptide was amplified by reverse transcription polymerase chain reaction (RT-PCR) method using isolated total RNA as template. The amplified cDNA fragment was inserted to plasmid pUC 18. The sequencing result shows that length of CGH cDN A fragment is 669bp. ORF include of 633bp, encoding 210 amino acids with molecular 23.6 KDa weight and pI is 6.28. CGH peptide include 12.4% Basic Amino Acids, 11.9% Acidic Amino Acids, 32.4% Hydrophilic Amino Acids, 43.3% Hydrophonic Amino Acids. By blasting the homologous sequences in GenBank databases, the sequence of grass carp GH cDNA from pituitary is 98% homologous compared with the previously cloned GH cDNA of grass carp .The CGH cDNA fragment was inserted into pGEX-4T-l to construct the expression plasmid. The recombinant plasmid was digested by BamH I and EcoR Ito identify whether the CGH cDNA fragment was inserted into the plasmid, the pGCGH was transformed into E.coli BL21 competent cells. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE. The results were found that the specific 670bp DNA bases of CGH was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 50 KDa which is consisted of a 23.6 KDa protein deduced from the CGH gene sequence and GST (26 KDa). These indicated that the grass carp GH gene was correctly transcribed and translated in the recombinant bacteria, and could be employed to further develop a novel growth-regulating reagents to promote the health development offish.