【摘要】：表皮生長因子受體(Epidermal Growth Factor Receptor,EGFR)屬于受體酪氨酸激酶,在哺乳動(dòng)物生長發(fā)育過程有重要的調(diào)節(jié)作用。EGFR通過激活mTOR信號(hào)通路調(diào)節(jié)脂肪酸和膽固醇的合成,脂肪酸和膽固醇合成相關(guān)基因反過來對(duì)EGFR也發(fā)揮調(diào)節(jié)作用。EGFR的研究熱點(diǎn)主要集中在腫瘤癌癥領(lǐng)域,而在脂肪酸代謝領(lǐng)域的研究較少,在反芻動(dòng)物如奶山羊和奶牛上更是鮮見類似報(bào)道。本研究通過克隆得到了山羊EGFR基因CDs區(qū)序列。原核表達(dá)截短型EGFR蛋白,制備了兔抗山羊EGFR多克隆抗體。成功包裝Ad-EGFR重組腺病毒并合成針對(duì)EGFR基因的siRNA序列,通過RT-qPCR檢測EGFR基因過表達(dá)和干擾后對(duì)山羊原代乳腺上皮細(xì)胞脂質(zhì)合成相關(guān)基因的影響,以期通過本研究為EGFR基因?qū)θ槌煞执x調(diào)控機(jī)理的研究奠定理論基礎(chǔ)。獲得的主要研究成果如下:(1)克隆得到奶山羊EGFR基因CDs區(qū)序列3 627 bp,編碼1 208個(gè)氨基酸。EGFR蛋白的第24和25個(gè)氨基酸之間為信號(hào)肽切割位點(diǎn),N端區(qū)域具有較強(qiáng)的疏水性,C端具有較強(qiáng)的親水性。奶山羊泌乳前期和泌乳后期EGFR基因的表達(dá)量最高,干奶期的表達(dá)量最低。(2)克隆得到截短型EGFR蛋白(ECD),并構(gòu)建pET-32a(+)-ECD原核表達(dá)載體,該載體轉(zhuǎn)化BL21大腸桿菌后37℃,0.8 mmol/L IPTG誘導(dǎo)6 h得到重組蛋白,重組蛋白純化效果良好。iELISA檢測發(fā)現(xiàn)抗血清效價(jià)高達(dá)1:16 000,Western blot檢測呈現(xiàn)較好的抗體特異性。(3)成功構(gòu)建并包裝得到重組腺病毒Ad-EGFR,感染山羊原代乳腺上皮細(xì)胞36 h,FASN、ACC、LXRα、LXRβ、SREBP1、SCD1、ACSS1、DGAT1、DGAT2和SP1基因表達(dá)量顯著上調(diào)(P0.05),FABP3基因表達(dá)量顯著下調(diào)(P0.05)。Western blot檢測發(fā)現(xiàn),感染重組腺病毒Ad-EGFR 36 h以上乳腺上皮細(xì)胞中EGFR蛋白顯著表達(dá),而且EGFR蛋白能夠被組成性激活。(4)成功合成針對(duì)EGFR基因的siRNA序列,轉(zhuǎn)染山羊原代乳腺上皮細(xì)胞24 h后通過RT-qPCR檢測發(fā)現(xiàn)ACSL1、DGAT2、PRLR和LF基因顯著上調(diào)(P0.05),SCD1、FASN、ACC、LXRα、LXRβ和SP1基因顯著下調(diào)(P0.05),而乳鐵蛋白的表達(dá)呈現(xiàn)上調(diào)趨勢(P0.05)。綜上所述:本研究成功克隆得到了EGFR基因的CDs區(qū)序列,并通過該序列原核表達(dá)出截短型EGFR蛋白,制備了兔抗山羊EGFR蛋白多克隆抗體。過表達(dá)和干擾EGFR基因后奶山羊乳腺上皮細(xì)胞中乳成分合成基因發(fā)生改變。
[Abstract]:Epidermal growth factor receptor (Epidermal Growth Factor Receptor,EGFR) is a receptor tyrosine kinase that plays an important role in mammalian growth and development. EGFR regulates the synthesis of fatty acids and cholesterol by activating the mTOR signaling pathway. Fatty acid and cholesterol biosynthesis related genes, in turn, play a regulatory role in EGFR. The research focus is mainly in the field of cancer, but less in the field of fatty acid metabolism. Similar reports are rare in ruminants such as dairy goats and cows. In this study, the CDs region of goat EGFR gene was cloned. Prokaryotic expression of truncated EGFR protein was used to prepare rabbit anti goat EGFR polyclonal antibody. Ad-EGFR recombinant adenovirus was successfully packaged and siRNA sequence for EGFR gene was synthesized. The effect of overexpression and interference of EGFR gene on lipid synthesis related genes in goat primary mammary epithelial cells was detected by RT-qPCR. The aim of this study was to lay a theoretical foundation for the study of the regulation mechanism of EGFR gene on milk composition metabolism. The main results obtained are as follows: (1) cloning of dairy goat EGFR gene CDs region 3 627 bp, encoding 1 208 amino acids .EGFR protein between the 24th and 25th amino acids is a signal peptide cleavage site with a strong hydrophobic region. The C terminal has strong hydrophilicity. The expression of EGFR gene was the highest in prelactation and late lactation, and the lowest in dry milk. (2) the truncated EGFR protein (ECD), was cloned and the prokaryotic expression vector of pET-32a () -ECD was constructed. The recombinant protein was obtained after the transformation of the vector into E. coli BL21 at 37 鈩，

本文編號(hào)：2251765