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Cloning and Expression of the Prolactin Gene and Its Physiological Activity in Shitou Goose

Author: WuHuiYing
Tutor: JiaRuMin
School: Guangdong Ocean University
Course: Animal Genetic Breeding and Reproduction
Keywords: Goose Prolactin (PRL) gene Broodiness behavior Soluble protein Restructuring PRL
CLC: S835
Type: Master's thesis
Year: 2010
Downloads: 75
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Abstract

Prolactin (Prolactin, PRL) is a peptide hormone secreted by the anterior pituitary eosinophils, and growth hormone (Growth hormone, GH) and placental lactogen (Placental Lactogens, PLs) belong to the same gene family, exists in almost All vertebrates. Prolactin has a wide range of physiological functions, PRL mother avian (bird) nest brooding behavior and regulation of reproductive activity of key hormones in avian (bird) class. On the nest during the PRL elevated expression levels inhibit the secretion of pituitary gonadotropins, to make poultry follicle stops growing and termination of egg. Nests behavior in the modern poultry industry has become an important limiting factor restricting poultry breeding performance and breeding efficiency. This study by cloning, expression of goose PRL gene sequence of the mature region prepared with the reorganization of the biological activity of PRL protein;, and PRL in goose regulatory role broodiness preliminary observation, for further research PRL biological activity in goose nests physiological regulatory mechanisms in the egg laid the foundation. (1) goose PRL gene cloning and sequence analysis using the Trizol reagents from the pituitary gland of goose extraction of total RNA, RT-PCR amplification of PRL gene sequences, cloned into pMD18-T vector measured nucleoside acid sequence homology with the release of PRL gene sequences in the GenBank comparison. The results show that the goose PRL gene 690 nucleotides, encoding 229 amino acids, and Sichuan white goose, northeast seed goose nucleotide sequence homology of 100%, compared to 99.9% with Eastern Zhejiang white geese The amino acid sequence homology in more than 99%. Bioinformatics analysis found the goose with other geese species PRL amino acid sequence has a potential signal peptide cleavage site (28VTS-LP32) and a heparin-binding sites, suggesting that the goose PRL protein secondary structure from multiple α helix section 70 to 76, 95 to 102,150 to 155 and 207 to 213 of the amino acid sequence of the N-terminal and β-turn and random coil form, its antigen epitopes advantages Area. Designed a pair (2) goose prokaryotic expression of recombinant PRL gene specific primers with restriction sites the mature PRL District fragment was amplified from the recombinant plasmid pMD18-T-PRL the double digested recovery target fragment through the same enzyme The cut processing pET32a () vector connection conversion E.coliDH5α, extracted recombinant plasmid PCR and restriction enzyme digestion, the recipient strain was transformed into E.coli BL21 (DE3). IPTG 37 ℃ induced expression of proteins at different times, by SDS-PAGE and Western Blot. The results showed PAGE gel and NC membrane molecular mass to approximately 41KDa specific protein band expression product as a soluble protein induced 4h expressed recombinant PRL fusion protein accounted for 53.6% of the total protein in bacteria, specifically with PRL antibody reaction. Induced 4 h, at different temperatures and found that the highest expression level of 37 ° C target protein. (3) recombinant PRL fusion protein purification and identification of antigenic take the pET32-PRL expression bacterial ultrasonic lysis supernatant by Ni2-NTA gel column affinity purification expression of the protein, followed by elution gradient concentration of imidazole solution unbound protein, and collecting the a PRL target protein. Expression products after SDS-PAGE gel separation by electron transfer to NC membrane by Western Blot with rabbit anti-chicken PRL-IgG and HRP-labeled goat anti-rabbit antibody reaction, DAB chromogenic results show that the expression of the target protein can with the the PRL antibody occurrence of specific binding reactions, molecular size of approximately 41KDa specific bands formed in the NC film that PRL gene product has a good antigenic. The above results for further study of the biological activity of PRL in the goose on the nest, and laid the foundation for physiological regulatory mechanisms in the egg. (4) recombinant PRL goose nest resistance of the recombinant prepared by mixing the antigen the the PRL protein with Freund's complete adjuvant, using different doses packet immune nest goose, setting the saline control group of incomplete Freund's adjuvant. The observed experimental group and the control group to nest regularly detect groups goose serum PRL levels. The results showed that the prepared recombinant geese PRL protein has a strong immunogenicity. The PRL of the immune high dose (4mg) can quickly reduce PRL levels in the circulating blood, can quickly and efficiently early in the immune suppression goose on nest behavior.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Poultry > Goose
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