A Novel Test Method Of Goose Prolactin Based On PRLR-JAK-STAT5 Signal Transduction Pathway

PRL(Prolactin, PRL) is a hormone that has a wide range of physiological functions. In birds, it plays as a key hormone that promotes female avian(bird) nest behavior occurrence and regulats reproductive behaviors. During the nest, the PRL expression was increased, and suppressed the secretion of pituitary gonadotropin, finally, poultry follicle development was hindered and maintian thebroodiness. In modern poultry industry, broody behavior has become an important limiting factor that restricting poultry breeding and rearing performance benefits. PRL is also one of the key hormones for the study of goose anti-season reproductive technology. Different concentrations of PRL exhibited different roles during the regulations in reproductive activity of birds. Therefore it is particularly necessary to accurate measuring the concentrations of PRL in serum. In order to develop a high sensitive method of detecting goose PRL, luciferase detecting system based on PRLR-JAK-STAT5 signal transduction pathway was designed in the present study.Firstly, in this experiment, RNA was isolated from the fresh ovary tissue samples of healthy egg-laying female goose, and the first strand of CDS was synthesized by reverse transcription PCR. The CDS region of goose PRLR gene was cloned by PCR using the, synthesized CDS as template and a 6x His tag was introduced at the end of the sequence, then the sequence was inserted into the eukaryotic expression vector pCMV6-Entryby HindIII and XhoI restriction sites, constructed as the recombinant vector pCMV6-PRLR-His-Entry, pCMV6-PRLR-His-Entry was transfected into HEK293 T cells transiently, after 30 h culture, the cells were haversted by collectedprotein of the cellsusing beyotime protein lysate buffer and then analyzed the expression of the recombinant PRLR-His protein in the cells by Western-blot.Secondly, the signal transducer and activator of transcription 5(Signal transducer and activator of transcription 5, STAT5) was artificially synthesized. And then the sequence as well as PRLR sequence were inserted into the luciferase reporter pGL3-Enhancer by KpnI and Xho I restriction sites and pCMV6-Entry vectors by HindIII and Xho I restriction sites respectively, both the recombinant vectors, pGL3-(STAT5)5-Enhancer and pCMV6-PRLR-Entry were used as signal responder vector. Then the two recombinant vectors along with the internal control pRL-TK vector were transfected into HEK293 T cells transiently. After 24 h culture, the transfected cells werestimulated by a serial of different concentrations of chicken PRL(0 ng/mL, 30 ng/mL, 60 ng/m L, 90 ng/mL and 120 ng/mL). After 24 h stimualtion, the relative activity of luciferase enzyme was detected by dual-luciferase detection system.Finally, the two recombinant vectors pGL3-(STAT5)5-Enhancer, pCMV6-PRLR-Entry along with the screening vector pEZX-MR03 containing the reporter gene EGFP and screening gene puromycin were linearized,and transfected into HEK293 T cells. After 30 d puromycin screening, stable transgenic monoclonal cell lines were isolated and expanded. The integration detection of pCMV6-PRLR-Entry and pGL3-(STAT5)5-Enhancer vectors in the genome of stable transgenic monoclonal cell lines by conventional PCR, using the extracted genomic DNA as template. Results showed that 10 stable transgene cell lines carry all the three vectors were isolated. Then the ten stable transgene cell lines stimulated by a serial of different concentrations of chicken PRL(0 ng/m L, 30 ng/mL, 60 ng/mL and 90 ng/m L). After 6 h stimulation, the RNA of ten stable transgene cell lines were extracted and the expression changes of luciferase gene were analyzed by quantitative real-time PCR(Quantitative real time PCR, QRT-PCR). The activity of luciferase enzyme wasalso detected by single-luciferase detection system. Results showed thatafter treated with different concentrations of chicken PRL, luciferase gene mRNA expression level was up regulated and the enzyme activity was also enhanced in dose dependent manner in one of these ten cell lines.These results demonstrated that it is feasible of detecting PRL bioactivity using the constructed PRLR-JAK-STAT5 signal transduction system and can be further used in measuring poultry PRL concentrations.