Cloning And Expression Of The Prolactin Gene And Its Physiological Activity In Shitou Goose

Prolactin (Prolactin, PRL) is a peptide hormone secreted by the pituitary acidophilic cell, along with the growth hormone (Growth hormone, GH) and placental prolactin (Placental Lactogens, PLs) belong to the same gene family, which was found in almost all vertebrates. Prolactin has a wide range of physiological functions in avian (bird), and PRL is a key hormone to promote the hatching on the nest incubating behavior and regulate the activities of reproduction. During the nesting period, the expression of the PRL increased which suppress the pituitary gonadotropin secretion, and the laying and follicle development subsequently stopped. In the modern poultry industry, the broodiness behavior had become an important limiting factor on constraint birds breeding performance and breeding efficiency. The present study was used to clone and express mature PRL gene sequence of the Shitou goose, then the purified recombinant PRL was prepared and the biological effect of PRL on nesting behavior of the Shitou goose was observed. The present study also laid scientific basis for further study of physiological regulation mechanism of PRL in broodiness and laying behavior of the Shitou goose.(1) The cloning and sequence analysis of Shitou goose PRL geneWith Trizol reagent, the total RNA was extracted from the Shitou goose's pituitary, PRL gene was amplified by RT-PCR, and the amplified segment was then cloned to pMD18-T vector. The nucleotide sequence of the PRL gene was determined, and the homology of nucleotide and amino acid sequence of PRL gene in Shitou goose and other avian breeds were analysed. The results showed that the Shitou goose PRL gene was consisted by 690 nucleotides coding for 229 amino acids, and the nucleotide sequence homology came to 99-100% among Shitou Goose, Sichuan Goose, Northeast Zi Goose and Zhedong Goose, the amino acid homology also came to more than 99%. Moreover, the PRL amino acid sequences of the Shitou goose and other goose species had a potential signal peptide splice site (28VTS-LP32) and a heparin binding site, suggesting that a number ofαhelix andβangle and random coil in PRL secondary structure, and the N-terminal amino acids such as 70 ~76, 95~102, 150~155 and 207~213 may be the main epitope domain in PRL by the bioinformatics analysis.(2) Expression of recombinant PRL of Shitou goose in E.coliA pair of specific primer with restriction enzyme site was designed to amplify the sequence of mature PRL peptide from pMD18-PRL recombinant plasmid, and the amplified fragment was then inserted into pET32a(+) expression vector, and the E.coli DH5αcompetent cells were transformed, and the recombinant plasmid pET32-PRL was extracted and identified by PCR and restriction enzyme digestion. Subsequently, the recombinant plasmid pET32-PRL was conduced into the E.coli BL21 (DE3) expression bacteria, then the recombinant PRL was expressed by IPTG induction at 37℃, and detected by SDS-PAGE and Western Blot. The results showed that the specific protein bands with molecular weight of 41KDa were appeared on both of the PAGE gel and NC membrane. The recombinant PRL fusion protein was soluble, accounting for 53.6% of total bacterial protein, and it can specifically react with PRL antibody.(3) Recombinant fusion PRL protein purification and antigenicityThe bacteria expressing recombinant PRL fusion protein was collected and lysised by ultrasonic, and supernatant was used to purify PRL protein by Ni2 +-NTA affinity gel. The purified recombinant PRL was then condensed and dried with vacuum freeze-drying system. The purified recombinant PRL was detected by SDS-PAGE and Western-blot. The result showed that a single protein band with molecular weight of 41KDa was observed on both of the PAGE gel and the NC membrane, indicating that the PRL gene expression products had good antigenicity. The results above laid the foundation for the further study of the biological activity of PRL and the physiological regulation mechanism on the broodiness and laying of Shitou goose.(4) The effect of the recombinant PRL on the broodiness Shitou gooseThe antigen were prepared by recombinant PRL proteins mixing with Freund's adjuvant, and the brooding geese were grouped and then immuned with different doses. The broodiness behavior of the geese was observed, and the serum PRL concentration was detected. The results indicated that the recombinant PRL protein showed strong immunogenicity, and the broodiness behavior of the Shitou goose was rapidly inhibited then the endogenous serum PRL level was accordingly decreased by high-dose PRL (4mg) immunization.