奶山羊表皮生长因子受体（EGFR）基因CDs区的克隆分析与功能的初步研究
发布时间：2018-10-04 20:27
【摘要】：表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)属于受体酪氨酸激酶,在哺乳动物生长发育过程有重要的调节作用。EGFR通过激活mTOR信号通路调节脂肪酸和胆固醇的合成,脂肪酸和胆固醇合成相关基因反过来对EGFR也发挥调节作用。EGFR的研究热点主要集中在肿瘤癌症领域,而在脂肪酸代谢领域的研究较少,在反刍动物如奶山羊和奶牛上更是鲜见类似报道。本研究通过克隆得到了山羊EGFR基因CDs区序列。原核表达截短型EGFR蛋白,制备了兔抗山羊EGFR多克隆抗体。成功包装Ad-EGFR重组腺病毒并合成针对EGFR基因的siRNA序列,通过RT-qPCR检测EGFR基因过表达和干扰后对山羊原代乳腺上皮细胞脂质合成相关基因的影响,以期通过本研究为EGFR基因对乳成分代谢调控机理的研究奠定理论基础。获得的主要研究成果如下:(1)克隆得到奶山羊EGFR基因CDs区序列3 627 bp,编码1 208个氨基酸。EGFR蛋白的第24和25个氨基酸之间为信号肽切割位点,N端区域具有较强的疏水性,C端具有较强的亲水性。奶山羊泌乳前期和泌乳后期EGFR基因的表达量最高,干奶期的表达量最低。(2)克隆得到截短型EGFR蛋白(ECD),并构建pET-32a(+)-ECD原核表达载体,该载体转化BL21大肠杆菌后37℃,0.8 mmol/L IPTG诱导6 h得到重组蛋白,重组蛋白纯化效果良好。iELISA检测发现抗血清效价高达1:16 000,Western blot检测呈现较好的抗体特异性。(3)成功构建并包装得到重组腺病毒Ad-EGFR,感染山羊原代乳腺上皮细胞36 h,FASN、ACC、LXRα、LXRβ、SREBP1、SCD1、ACSS1、DGAT1、DGAT2和SP1基因表达量显著上调(P0.05),FABP3基因表达量显著下调(P0.05)。Western blot检测发现,感染重组腺病毒Ad-EGFR 36 h以上乳腺上皮细胞中EGFR蛋白显著表达,而且EGFR蛋白能够被组成性激活。(4)成功合成针对EGFR基因的siRNA序列,转染山羊原代乳腺上皮细胞24 h后通过RT-qPCR检测发现ACSL1、DGAT2、PRLR和LF基因显著上调(P0.05),SCD1、FASN、ACC、LXRα、LXRβ和SP1基因显著下调(P0.05),而乳铁蛋白的表达呈现上调趋势(P0.05)。综上所述:本研究成功克隆得到了EGFR基因的CDs区序列,并通过该序列原核表达出截短型EGFR蛋白,制备了兔抗山羊EGFR蛋白多克隆抗体。过表达和干扰EGFR基因后奶山羊乳腺上皮细胞中乳成分合成基因发生改变。
[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，
本文编号：2251765
[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，
本文编号：2251765
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