基于PRLR-JAK-STAT5信号传导通路测定鹅催乳素的新方法

A Novel Method of Detecting Goose Prolactin Based on PRLR-JAK-STAT5 Signal Transduction Pathway

旨在建立鹅催乳素(Goose prolactin,gPRL)的高灵敏度的测定方法。本研究设计出基于PRLR-JAKSTAT5信号通路的荧光素酶报告基因系统。首先克隆鹅PRLR基因CDS区序列,合成信号转导和转录激活因子5(Signal transducer and activator of transcription 5,STAT5)信号应答序列,并分别插入真核表达载体pCMV6-Entry和荧光素酶报告载体pGL3-Enhancer中,构建成为信号接收载体和信号应答载体。然后将两载体同筛选基因载体pEZX-MR03及内参载体pRL-TK共转染到HEK293T细胞中,经嘌呤霉素筛选后获得稳定转染的转基因细胞株。分别用终浓度为0、30、60、90ng·mL^(-1)的PRL刺激转基因细胞,通过qRT-PCR和双荧光素酶检测系统测定在不同PRL浓度下转基因细胞株中荧光素酶(Luciferase,Luc)基因的相对表达量和相对活性的变化。筛选出10株成功整合有全部4个转基因载体的转基因细胞株,经过不同浓度PRL刺激后,筛选出一株细胞,其荧光素酶基因表达量和酶活性均表现出随PRL浓度升高而上调的趋势。结果表明,建立的基于PRLR-JAK-STAT5信号传导系统检测鹅PRL生物活性的新方法是可行的,为准确测定家禽PRL奠定基础。

In order to develop a high sensitive method of detecting goose prolactin,luciferase detecting system based on PRLR-JAK-STAT5 signal transduction pathway was designed in the present study.Firstly the CDS region of goose PRLRgene was cloned,the signal transducer and activator of transcription 5（STAT5）was artificially synthesized and inserted into the eukaryotic expression vector pCMV6-Entry and luciferase reporter pGL3-Enhancer plasmid as signal receiver vector and signal responder vector respectively.Secondly,the two recombinant vectors along with puromycin screening vector pEZX-MR03 and internal control pRL-TK vector were transfected into HEK293 Tcells.After puromycin screening,stable transgenic cell lines were isolated and stimulated by a serial of different concentrations of chicken PRL（0,30,60,90ng·mL（-1））.Finally the mRNA relative expression level and enzyme activity of luciferase enzyme were detected by qRTPCR and dual-luciferase detection system.Results showed that 10 stable transgene cell lines carried the 4vectors were isolated.After treated with different concentrations of chicken PRL,lucif-erase gene mRNA expression level was up regulated and luciferase enzyme activity also enhanced in dose dependent manner in one of these 10 cell lines.These results demonstrated that it was feasible of detecting PRL bioactivity using the constructed PRLR-JAK-STAT5 signal transduction system and could be further used in measuring poultry PRL concentrations.