绵羊EIF3L、EIF3H、EEF1D基因克隆表达及对羊毛生长相关因子的影响

发布时间：2018-03-28 11:16

  本文选题：真核翻译起始因子　切入点：真核翻译延伸因子　出处：《塔里木大学》2017年硕士论文

【摘要】：羊毛的生长发育受多种因素的影响,其生长发育过程和周期性生理调控对羊毛性状相关基因的测定及调控网络的探索有着积极的影响。真核翻译起始因子3L(EIF3L)、真核翻译起始因子3H(EIF3H)、真核翻译延伸因子(EEF1D)是一类在细胞内参与调控蛋白质翻译的细胞因子,与细胞活动紧密相关。本研究首先以中国美利奴羊成纤维细胞总RNA为模板,用RT-PCR扩增获得EIF3L、EIF3H、EEF1D基因mRNA的CDS区编码序列,通过酶切及纯化后,分别连接至原核表达载体pet28α(+)、真核表达载体pcDNA3.1(-),构建EIF3L、EIF3H、EEF1D基因的原核表达载体和真核表达载体。测序结果显示,获得的目的基因序列与Genebank上绵羊预测序列相似性分别为100%、99.91%、100%,其中EIF3H序列第1055bp位置上的核苷酸碱基由A突变为G,编码的氨基酸由天冬酰胺(Asn/N)突变为AGC丝氨酸(Ser/S);对序列比对分析发现,EIF3L、EIF3H、EEF1D核苷酸序列及编码的氨基酸与人、牛、鼠、猪、兔等动物的核苷酸序列及编码的氨基酸序列有一定的同源性。将重组的原核表达质粒转入E.coli BL21(DE3)后,在37℃,终浓度为0.75mmol·L~(-1)的IPTG诱导3.5 h后,分别产生了以包涵体形式表达的蛋白,其中EIF3L蛋白分子量大小约为69kDa,EIF3H蛋白分子量大小约为40kDa,EEF1D蛋白分子量大小约为40kDa,经Western blot检测该蛋白分子具有很好的反应原性。将构建好的重组真核表达载体分别瞬时转染至原代培养的中国美利奴羊成纤维中,再将转染细胞分别提取总RNA和总蛋白,进行RT-qPCR和Western blot检测后,结果显示,EIF3L、EIF3H的mRNA表达量和蛋白表达量相对于正常的成纤维细胞均有不同程度的上调,EEF1D的mRNA表达量未出现明显上调,蛋白表达量升高。在与羊毛相关基因表达量检测中发现,转染重组真核表达载体的成纤维细胞mRNA表达量分别有着不同程度的上调和下调。采用RT-qPCR和Western blot对中国美利奴羊和哈萨克羊进行组织表达谱检测,结果显示,EIF3L、EIF3H、EEF1D的mRNA和蛋白在中国美利奴羊和哈萨克羊的心脏、肝脏、脾脏、肺脏、肾脏、皮肤、肌肉组织中均有不同程度的表达且具有一定差异。
[Abstract]:The growth and development of wool are affected by many factors. Its growth and development process and periodic physiological regulation have a positive effect on the determination of the genes related to wool traits and the exploration of regulatory networks. The eukaryotic translation initiation factor 3L, the eukaryotic translation initiation factor 3H, the eukaryotic translation initiation factor 3H, the eukaryotic translation extension factor EEF1D). Is a class of cytokines involved in the regulation of protein translation in cells. In this study, the total RNA of Chinese Merino sheep fibroblasts was used as a template to amplify the coding sequence of the CDS region of the EIF3L- EIF3HIF3HEEEF1D gene mRNA, which was digested and purified by enzyme digestion. The prokaryotic expression vector and eukaryotic expression vector of EIF3L- EIF3HEEF1D gene were constructed by ligating to the prokaryotic expression vector pet28 伪 (pet28 伪) and eukaryotic expression vector pcDNA3.1HEEF1D, respectively. The similarity between the obtained target gene sequence and the predicted sequence of sheep on Genebank is 100 ~ 99.91%, in which the nucleotide base at the 1055bp position of the EIF3H sequence is mutated from A to G, and the encoded amino acid is mutated from asparagine Asn / N to AGC serine serine serine. The nucleotide sequence and amino acid encoding of EIF3L, EIF3H, EEF1D and human were found by comparison analysis. The nucleotide sequence and amino acid sequence of bovine, mouse, pig, rabbit and so on have some homology. After the recombinant prokaryotic expression plasmid was transferred into E.coli BL21 (DE3), the recombinant plasmid was induced by IPTG at 37 鈩，

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