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  本文選題：鵝催乳素受體 + 信號轉(zhuǎn)導��； 參考：《山東農(nóng)業(yè)大學》2016年碩士論文

【摘要】：催乳素(Prolactin,PRL)是具有廣泛生理功能的一種激素。在禽(鳥)類中,是促進母禽(鳥)就巢行為發(fā)生和調(diào)控生殖活動的關(guān)鍵激素之一。就巢期PRL表達量升高抑制垂體促性腺激素的分泌,使得禽類卵泡停止發(fā)育并終止產(chǎn)蛋,從而形成固定的就巢行為。禽類就巢行為已成為現(xiàn)代養(yǎng)禽業(yè)中制約禽類繁殖性能和飼養(yǎng)效益提高的重要限制因素。PRL也是研究鵝反季節(jié)繁殖技術(shù)中的關(guān)鍵激素之一。不同濃度PRL對禽類生殖活動的調(diào)控表現(xiàn)出不同的作用,因此,準確測定不同繁殖周期中血漿PRL濃度尤為必要。為準確測定鵝PRL(goose PRL,gPRL)濃度,本研究設計了基于PRLR-JAK-STAT5信號通路測定gPRL的新方法。本試驗首先從健康產(chǎn)蛋鵝卵巢組織提取核糖核酸(ribonucleic acid,RNA),RT-PCR技術(shù)克隆了鵝催乳素受體基因(Prolactin receptor,PRLR)CDS區(qū)序列,并在該序列末尾添加6xHis標簽,將加標簽后的序列插入到真核表達載體pCMV6-Entry的Hind III和XhoI酶切位點中,構(gòu)建成為重組載體pCMV6-PRLR-His-Entry,并將其瞬時轉(zhuǎn)染到HEK293T細胞中,培養(yǎng)30 h后,用碧云天蛋白裂解液裂解經(jīng)轉(zhuǎn)染的HEK293T細胞并收集細胞內(nèi)蛋白質(zhì),利用Western-blot驗證轉(zhuǎn)染的pCMV6-PRLR-His-Entry中重組PRLR-His蛋白在HEK293T細胞中的表達。然后人工合成了信號轉(zhuǎn)導和轉(zhuǎn)錄激活因子5(Signal transducer and activator of transcription 5,STAT5)信號應答序列。同時將鵝PRLR基因CDS區(qū)序列和STAT5序列分別插入到真核表達載體pCMV6-Entry和熒光素酶報告載體pGL3-Enhancer的KpnI和XhoI酶切位點中,構(gòu)建成為信號接收載體pCMV6-PRLR-Entry和信號應答載體pGL3-(STAT5)5-Enhancer。然后將上述兩載體同內(nèi)參載體pRL-TK瞬時轉(zhuǎn)染到HEK293T細胞中,培養(yǎng)24 h后加PRL至終濃度分別為0 ng/mL、30 ng/m L、60 ng/m L、90 ng/m L和120ng/m L,繼續(xù)培養(yǎng)24 h并通過雙熒光素酶檢測系統(tǒng)測定細胞中熒光素酶(Luciferase,Luc)相對活性的變化。將pCMV6-PRLR-Entry和pGL3-(STAT5)5-Enhancer同含有增強綠色熒光蛋白(Enhanced Green Fluorescent Protein,EGFP)報告基因和嘌呤霉素篩選基因的載體pEZX-MR03均線性化后共轉(zhuǎn)染到HEK293T細胞中,經(jīng)嘌呤霉素篩選30 d后,挑取單克隆細胞,并擴大培養(yǎng),獲得轉(zhuǎn)基因單克隆細胞株。提取轉(zhuǎn)基因單克隆細胞株基因組脫氧核糖核酸(Deoxyribonucleic acid,DNA),通過普通PCR分別驗證pCMV6-PRLR-Entry和pGL3-(STAT5)5-Enhancer在轉(zhuǎn)基因細胞株基因組中的穩(wěn)定整合,共篩選出10株成功整合有全部轉(zhuǎn)基因載體的穩(wěn)定轉(zhuǎn)染單克隆細胞株。擴大培養(yǎng)篩選出的成功整合有pCMV6-PRLR-Entry和pGL3-(STAT5)5-Enhancer的單克隆細胞株,添加PRL至終濃度分別為0 ng/m L、30 ng/m L、60 ng/mL和90ng/m L,繼續(xù)培養(yǎng)6 h后提取細胞RNA,通過熒光實時定量PCR(Quantitative real time PCR,QRT-PCR)測定轉(zhuǎn)基因細胞株中Luc基因相對表達量的變化,同時通過單熒光素酶檢測系統(tǒng)測定單克隆細胞中Luc的活性,最終篩選出一株細胞,其Luc基因相對表達量及酶活性表現(xiàn)出隨PRL濃度升高而明顯上調(diào)的趨勢。結(jié)果證明本研究建立的基于PRLR-JAK-STAT5信號傳導系統(tǒng)檢測具生物活性gPRL濃度的新方法是可行的,為鵝及其他家禽PRL生物活性測定方法奠定基礎(chǔ)。
[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.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S835

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