Cloning Of PRL-cDNA, Tissue Distribution In Southern Catfish And Construction Of Prokaryotic Expression Vector

From March, 2006 to March, 2008, the full length of southern catfish PRL-cDNA was cloned with the methods of RT-PCR and RACE, and the PRL tissue distribution was detected in the southern catfish extrapituitary tissue with the method of RT-PCR, as well as prokaryotic expression vector of PRL was successfully to be constructed.The full length of the southern catfish PRL-cDNA is 1235bp, including 5'-untranslated region (90bp), open reading frame (639bp) and 3'-untranslated region (506bp). There is a conservative AATAAA polyadenylation signal was located 22 bases upsteam of the poly (A) tail formed with 17bp. The open reading frame codes 212 amino acids (aa). Its molecular weight is about 23 kDa, and its isoelectric point (pI) is 8.19 which were predictioned by the Vector NTI SUITE 9 programme of Informax 2003. The precurse of PRL includes a putative signal peptide and a mature peptide of 186 aa residues was predicted by signalP in which there are four Cys residues to form two disulfide bonds. After the precurse was synthesized, the signal peptide was cleaved and the mature peptide was released.The putative amino acids similarity analysis of southern catfish PRL indicated: the similarity between the southern catfish PRL and stinging catfish's was 89.2%, and the similarity between the southern catifish PRL and channel catfish's was 91.5%. The similarity between the southern catfish PRL and the other teleosts' is 51.0%-77.6%, and then compared with amphibians', reptile's, aves', and mammalia's is 27.9%-31.6%. And the similarity between the southern catfish PRL and human PRL is 29.1%. Phylogenetic analysis suggested that the southern catfish PRL, stinging catfish and channel catfish were clustered in one group, and formed a monophyletic group. The result is identical with the expectable result.The RT-PCR, the PRL gene expression result in about 20 tissues included every part of the brain, every stion of the digestive tract, liver, pancreatic gland, spleen, kidney, gonad, heart, skin, muscle and pituitary indicated: PRL gene was expressed highest in pituitary ,then testis, ovary, telencephalon, diencephalons, mesencephalon, cerebellum, medulla oblongata and olfactory sac, and expressed low level in liver, pancreatic gland, spleen, kidney, head kidney, heart and muscle, but was not expressed in cardiac, helicobacter, gastric, foregut, midgut, hindgut. The difference between this study and exist researches is that more tissues was used to study tissue expression of PRL, and each organ was divided into different parts more exactly than ever before. On this basis, the southern catfish PRL gene expression was detected in olfactory sac at the frist time which suggested that PRL will have some new functions to be found. It can provide new clue for the hot issue of PRL function research.On the base of PRL-cDNA full length of sequence, the sequencing result proved by TA cloning and sequencing indicated that the amplified sequence is the target sequence which coded the mature peptide of the southern catfish PRL through designing primer with enzymatic digestive site and PCR amplified the target sequence of the mature peptide of southern catfish PRL. Double enzymatic digested the recombinant T plasmid and expression vector pET-28, and purified the target sequence and pET-28 with gel extraction ,and linked the target fragment and pET-28 with T₄ ligase to form recombinant plasmid pET-28-PRL, then transformed plasmid pET-28-PRL into competent bacteria DH5α.,all of these processes constructed the recombinant plasmid pET-28-PRL successfully which expressed PRL in vitro , then the recombinant plasmid pET-28-PRL was transformed into competent BL₂₁. The successful construction of prokaryotic expression vector provided a basic work for further study physiologic function and application on the aquaculture of the southern catfish PRL.