Study On Cloning, Prokaryotic Expression And Biological Activity Of Growth Hormone Gene Of Schizothorax Prenanti

The cDNA encoding growth hormone(GH) peptide was amplified by reverse transcription polymerase chain reaction(RT-PCR),based on the total RNA isolated from the pituitary of Schizothorax prenanti.The amplified cDNA was inserted into plasmid pMD18-T.The sequencing result showed that the length of SGH cDNA fragment is 633bp,and the ORF included of 633bp,encoding 210 amino acids with molecular mass of 23.55 Kda and pI of 5.46. The SGH peptide included 10.7%basic amino acids,12.38%acidic amino acids,28.57% hydrophilic amino acids,36.7%hydrophobicity amino acids.By blasting the homologous sequences in GenBank database,the sequence of Schizothoraxprenanti GH from pituitary is 92%-97%homologous compared with the previously cloned GH of other fish in the same family.The SGH cDNA fragment was inserted into pET-32a(+) to construct the expression plasmid, and the recombinant plasmid was identified by PCR and digested with EcoRâ… and Xhoâ… ,and the positive recombinant plasmid was named as pET-mSGH.Then the pET-mSGH was transformed into E.coli BL21 competent cells.The mRNA and expressed protein were assayed by SDS-PAGE.The results showed that a new protein band was found in SDS-PAGE with molecular mass of about 41 KDa.These indicated that the Schizothorax prenanti GH gene was correctly transcribed and translated in the recombinant bacteria.Throught animal experiment,it is demonstrated that the test fishes was higer in growth rate of body weight and stem length,respectively 20.82%and 17.35%higer than control group, additionally,the appetition of test fishes was enhanced obviously,and the protein was proved to be biological active consequently.Conclusively,this study has established substantial bases for further use of GH,eukaryotic expression of GH,and developing new genetic praeparatum regulating the growth of fish.