基于PRLR-JAK-STAT5信号传导通路测定鹅催乳素的新方法研究

发布时间：2018-06-30 01:22

本文选题：鹅催乳素受体 + 信号转导　；参考：《山东农业大学》2016年硕士论文

【摘要】：催乳素(Prolactin,PRL)是具有广泛生理功能的一种激素。在禽(鸟)类中,是促进母禽(鸟)就巢行为发生和调控生殖活动的关键激素之一。就巢期PRL表达量升高抑制垂体促性腺激素的分泌,使得禽类卵泡停止发育并终止产蛋,从而形成固定的就巢行为。禽类就巢行为已成为现代养禽业中制约禽类繁殖性能和饲养效益提高的重要限制因素。PRL也是研究鹅反季节繁殖技术中的关键激素之一。不同浓度PRL对禽类生殖活动的调控表现出不同的作用,因此,准确测定不同繁殖周期中血浆PRL浓度尤为必要。为准确测定鹅PRL(goose PRL,gPRL)浓度,本研究设计了基于PRLR-JAK-STAT5信号通路测定gPRL的新方法。本试验首先从健康产蛋鹅卵巢组织提取核糖核酸(ribonucleic acid,RNA),RT-PCR技术克隆了鹅催乳素受体基因(Prolactin receptor,PRLR)CDS区序列,并在该序列末尾添加6xHis标签,将加标签后的序列插入到真核表达载体pCMV6-Entry的Hind III和XhoI酶切位点中,构建成为重组载体pCMV6-PRLR-His-Entry,并将其瞬时转染到HEK293T细胞中,培养30 h后,用碧云天蛋白裂解液裂解经转染的HEK293T细胞并收集细胞内蛋白质,利用Western-blot验证转染的pCMV6-PRLR-His-Entry中重组PRLR-His蛋白在HEK293T细胞中的表达。然后人工合成了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。同时将鹅PRLR基因CDS区序列和STAT5序列分别插入到真核表达载体pCMV6-Entry和荧光素酶报告载体pGL3-Enhancer的KpnI和XhoI酶切位点中,构建成为信号接收载体pCMV6-PRLR-Entry和信号应答载体pGL3-(STAT5)5-Enhancer。然后将上述两载体同内参载体pRL-TK瞬时转染到HEK293T细胞中,培养24 h后加PRL至终浓度分别为0 ng/mL、30 ng/m L、60 ng/m L、90 ng/m L和120ng/m L,继续培养24 h并通过双荧光素酶检测系统测定细胞中荧光素酶(Luciferase,Luc)相对活性的变化。将pCMV6-PRLR-Entry和pGL3-(STAT5)5-Enhancer同含有增强绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)报告基因和嘌呤霉素筛选基因的载体pEZX-MR03均线性化后共转染到HEK293T细胞中,经嘌呤霉素筛选30 d后,挑取单克隆细胞,并扩大培养,获得转基因单克隆细胞株。提取转基因单克隆细胞株基因组脱氧核糖核酸(Deoxyribonucleic acid,DNA),通过普通PCR分别验证pCMV6-PRLR-Entry和pGL3-(STAT5)5-Enhancer在转基因细胞株基因组中的稳定整合,共筛选出10株成功整合有全部转基因载体的稳定转染单克隆细胞株。扩大培养筛选出的成功整合有pCMV6-PRLR-Entry和pGL3-(STAT5)5-Enhancer的单克隆细胞株,添加PRL至终浓度分别为0 ng/m L、30 ng/m L、60 ng/mL和90ng/m L,继续培养6 h后提取细胞RNA,通过荧光实时定量PCR(Quantitative real time PCR,QRT-PCR)测定转基因细胞株中Luc基因相对表达量的变化,同时通过单荧光素酶检测系统测定单克隆细胞中Luc的活性,最终筛选出一株细胞,其Luc基因相对表达量及酶活性表现出随PRL浓度升高而明显上调的趋势。结果证明本研究建立的基于PRLR-JAK-STAT5信号传导系统检测具生物活性gPRL浓度的新方法是可行的,为鹅及其他家禽PRL生物活性测定方法奠定基础。
[Abstract]:Prolactin (PRL) is a hormone with extensive physiological functions. In the bird (bird) class, it is one of the key hormones that promote the occurrence and regulation of reproductive activities by the mother bird (bird). The increase in the expression of PRL in the nest stage inhibits the secretion of pituitary gonadotropin, causes the fowl follicles to stop developing and terminates the laying of eggs, thus forming a fixed position. Nest behavior. The behavior of poultry nesting has become an important restriction factor that restricts the reproductive performance and raising efficiency of poultry in modern poultry industry,.PRL is also one of the key hormones in the study of the anti seasonal reproductive technology of goose. The different concentrations of PRL in the regulation of poultry reproduction are different. Therefore, the accurate determination of different reproductive cycles is made. The concentration of plasma PRL is particularly necessary. In order to accurately determine the concentration of goose PRL (goose PRL, gPRL), this study designed a new method for the determination of gPRL based on PRLR-JAK-STAT5 signaling pathway. This experiment first extracted the ribonucleic acid (ribonucleic acid, RNA) from the healthy egg laying goose ovary tissue (ribonucleic acid, RNA), and the RT-PCR technique cloned the goose prolactin receptor gene (Prolactin). R) CDS region sequence, and add the 6xHis tag at the end of the sequence, insert the labeled sequence into the Hind III and XhoI enzyme sites of eukaryotic expression vector pCMV6-Entry, construct it as a recombinant vector pCMV6-PRLR-His-Entry, and instantaneously transfect it into the HEK293T cell, and after culture 30 h, the transfection HEK2 by the lysate lysate of the blue cloud sky protein lysate The 93T cells and the intracellular protein were collected, and Western-blot was used to verify the expression of the recombinant PRLR-His protein in the transfected pCMV6-PRLR-His-Entry. Then the signal transduction and transcription activator 5 (Signal transducer and activator of transcription 5, STAT5) signal response sequence was synthesized. The sequence and STAT5 sequence were inserted into the KpnI and XhoI loci of the eukaryotic expression vector pCMV6-Entry and the luciferase reporter pGL3-Enhancer, which were constructed to become signal carrier pCMV6-PRLR-Entry and pGL3- (STAT5) 5-Enhancer. of signal response carrier and then transfected the two carrier with the internal reference carrier pRL-TK to HEK293T fine. In the cell, after 24 h, the concentration of PRL to final concentration was 0 ng/mL, 30 ng/m L, 60 ng/m L, 90 ng/m L and 120ng/m L, and 24 h was continued and the relative activity of luciferase was determined by double luciferase detection system. D Green Fluorescent Protein, EGFP) the carrier pEZX-MR03 of the gene and the purinamycin screening gene was transfected into HEK293T cells. After screening 30 d by purinamycin, the monoclonal cell was selected and the McAb was expanded to obtain the genome deoxyribonucleic acid (Deo) of the transgenic monoclonal cell line (Deo). Xyribonucleic acid, DNA), the stable integration of pCMV6-PRLR-Entry and pGL3- (STAT5) 5-Enhancer in the genome of transgenic cell lines was verified by common PCR, and 10 stable transfection monoclonal cell lines with all transgenic carriers were successfully integrated. The successful integration of the expanded culture sieve was pCMV6-PRLR-Entry and pGL3- (STAT5) 5. The PRL to final concentration of -Enhancer was 0 ng/m L, 30 ng/m L, 60 ng/mL and 90ng/m L respectively. The cell RNA was extracted after 6 h, and the relative expression of the gene was measured by fluorescence real-time quantitative PCR. The activity of Luc in the monoclonal cells was determined, and a cell was screened out. The relative expression of Luc gene and the activity of the enzyme showed a tendency to rise obviously with the increase of PRL concentration. The result proved that the new method based on the PRLR-JAK-STAT5 signal transduction system to detect the bioactive gPRL concentration based on the PRLR-JAK-STAT5 signal transduction system is feasible, for geese and other families The basis of the determination of biological activity of avian PRL was laid.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S835

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