CDNA Clone And Prokaryotic Expression Of GH And PRL Genes Of Trachinotus Blochii

Growth hormone and prolactin are derived from the same ancestral gene,which is an important factor in regulating the growth and endocrine of fishes.This thesis clones and gains the ORF cDNA sequences of Trachinotus blochii growth hormone and prolactin so as to build up the prokaryotic expression vectors of growth hormone and prolactin.First,the author gains 887bp growth hormone cDNA sequences from the Transcriptome Sequencing.Based on the RT-PCR method,the author clones and obtains the growth hormone ORF cDNA sequences from the pituitary of Trachinotus blochii,and this ORF consists of 17 signal peptides and 162 mature peptides.The relative molecular weight of amino acid of this Trachinotus blochii growth hormone ORF is 22695.03,and its theoretical PI is 6.73,presenting partial acid,and its hydrophilic amino acid are 71.According to the comparison of amino acid sequences of ORF,the amino acid homology of Trachinotus blochii and fishes is more than 50%,of which the amino acid homology of Seriola lalandi of Carangidae is the highest,amounting to 89.6%.The ORF amino acid sequences of Trachinotus blochii growth hormone contain two label sequences of GH family:CnSdyliSPidkheTqrssvlkLlsis cqLveSW and CFKkDMhKVEtYltVakC.The GH protein of Trachinotus blochii is hydrophilic,and its third class structure has four alpha helices columns,which is similar to the GH protein structure of other fishes'.Second,through Trachinotus blochii Transcriptome Sequencing,the author gains1586 cDNA sequences.Based on the RT-PCR method,the author clones and gets ORF cDNA sequences of prolactin from the pituitary of Trachinotus blochii.This ORF includes 24 signal peptides and polypeptide chains consisting of 185 mature peptides.The relative molecular weight of amino acid of Trachinotus blochii prolactin ORF is 23178.89,and its theoretical PI is 8.60,of which the hydrophilic amino acid is 68.According to the analysis of ORF amino acid sequences,the similarity of amino acid sequences between Trachinotus blochii and other fishes is more than 55%,but only the homology of Sebastes schlegelii?Epinephelus coioides of Percifonnes is above 80%.The PRL ORF amino acid sequences of Trachinotus blochii contain two label sequences of PRL family:ChTssLqTPndkaqAlqlseadL mslarsLlqAW and CLRrDShKIDsFlkIlrC.The PRL protein of Trachinotus blochii is hydrophilic,and its third class structure has four alpha helices columns,which is similar to other fishes' PRL protein structure.The homology between growth hormone ORF amino acid sequences of Trachinotus blochii and its prolactin ORF amino acid sequences is 88%.Third,the pEASY-Blunt E1 Expression Vector constructs recombinant plasmid with the Trachinotus blochii GH and ORF cDNA sequences of PRL.Through observing cell transformation with BL21(DE3)and inducing the protein expression with IPTG,we can see from the SDS-PAGE that the PRL induction group in the position of 23.1KDa has clear protein bands,which is in accordance with the expected protein relative molecular weight of Trachinotus blochii PRL,and both the GH protein non-induction group and the induction group in the position of 23KDa have clear protein bands.