本文選題：山羊乳腺上皮細(xì)胞　切入點(diǎn)：胰島素誘導(dǎo)基因(INSIG)　出處：《畜牧獸醫(yī)學(xué)報》2016年09期

【摘要】：本研究旨在通過構(gòu)建西農(nóng)薩能奶山羊胰島素誘導(dǎo)基因1(INSIG1)的重組腺病毒(Adenovirus,Ad)超表達(dá)載體,研究該基因超表達(dá)后對乳腺上皮細(xì)胞中脂質(zhì)合成的影響,從而為該基因在山羊乳腺脂質(zhì)合成調(diào)控中的功能研究奠定基礎(chǔ)。根據(jù)GenBank(登錄號:JQ665439)中山羊INSIG1基因序列設(shè)計引物,PCR擴(kuò)增得到其CDS區(qū)序列。將目的基因連接到穿梭載體pAdTrack-CMV后獲得pAdTrack-CMV-INSIG1質(zhì)粒,將該質(zhì)粒PmeⅠ線性化后轉(zhuǎn)入大腸桿菌BJ5183感受態(tài)細(xì)胞進(jìn)行同源重組,獲得pAdEasy-INSIG1重組腺病毒超表達(dá)載體。該重組質(zhì)粒經(jīng)PacⅠ線性化后轉(zhuǎn)染293A細(xì)胞進(jìn)行病毒的包裝及擴(kuò)繁,利用LaSRT法測定其滴度。將獲得的重組腺病毒AdINSIG1感染山羊原代乳腺上皮細(xì)胞,利用實(shí)時熒光定量PCR檢測INSIG1及脂質(zhì)合成相關(guān)基因的mRNA表達(dá)情況,利用GPO-Trinder酶學(xué)反應(yīng)檢測細(xì)胞中甘油三酯的含量。結(jié)果表明,成功構(gòu)建了奶山羊INSIG1基因重組腺病毒超表達(dá)載體,獲得了具有較高滴度(2×108 U·mL-1)的超表達(dá)腺病毒Ad-INSIG1;Ad-INSIG1感染山羊原代乳腺上皮細(xì)胞48h后,與Ad-GFP組相比,INSIG1基因的mRNA表達(dá)量上調(diào)約500倍,固醇調(diào)節(jié)元件結(jié)合蛋白1(SREBP1)和SREBP裂解活化蛋白(SCAP)的mRNA表達(dá)量無明顯變化,參與脂肪酸從頭合成(ACCα、FASN)及去飽和(SCD1)基因的mRNA表達(dá)量均顯著下降(P0.05);參與甘油三酯合成的3個關(guān)鍵基因中,GPAM及DGAT2的mRNA表達(dá)量顯著下降(P0.05),AGPAT6的表達(dá)無顯著變化;同時,催化甘油三酯分解(ATGL)的基因mRNA表達(dá)量也顯著下降(P0.05);細(xì)胞內(nèi)甘油三酯含量無顯著變化。綜上所述,在山羊原代乳腺上皮細(xì)胞中,INSIG1能夠抑制脂質(zhì)合成相關(guān)基因的表達(dá),對山羊乳腺脂質(zhì)合成具有調(diào)控作用。
[Abstract]:The aim of this study was to construct a recombinant adenovirus Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Adenovirus-Ad@@Therefore, the study of the function of the gene in the regulation of lipid synthesis of goat mammary gland was laid a foundation.According to the goat INSIG1 gene sequence of GenBank (accession number: JQ665439), primers were designed to amplify the CDS region.The target gene was ligated into the shuttle vector pAdTrack-CMV to obtain the pAdTrack-CMV-INSIG1 plasmid. The plasmid Pme I was linearized and transferred into E. coli BJ5183 receptive cells for homologous recombination to obtain pAdEasy-INSIG1 recombinant adenovirus superexpression vector.The recombinant plasmid was linearized by Pac 鈪，

本文編號：1705942