肌肉特异性启动子IGF2表达载体构建及对牛骨骼肌卫星细胞增殖的影响

发布时间：2018-05-02 02:49

本文选题：IGF2 + 肌肉特异性启动子　；参考：《东北农业大学》2015年硕士论文

【摘要】：IGF2(胰岛素样生长因子2)是一种蛋白质,它由67个氨基酸残基组成,在个体的生长发育过程中起重要作用,是骨骼肌生长发育的重要调控因子。近年来,对IGF2的研究逐渐深入,但是通过过表达IGF2的方式来培育高产、生长发育快的优质肉牛方面的报道较少。本研究旨在获得带有高效特异性启动子的IGF2表达载体,研究其对牛骨骼肌卫星细胞(简称MDSCs)增殖的影响。欲提高IGF2在肌肉组织中的表达效率,首先需要获得一个含有IGF2的高效表达的载体,其中启动子的选择是一个重要因素。目前,巨细胞病毒(CMV)启动子是外源基因在真核细胞中表达常选用的启动子之一,该启动子非常高效,但其几乎不具有组织特异性,出于转基因生物安全方面的考虑,不能将其应用于转基因肉牛的生产中。所以,选取特异高效的启动子对于外源基因在真核细胞中的表达是必要的,本研究应用的是实验室前期构建的具有特异性且活性相对较高的三种启动子:Desminpro(300)是牛结蛋白基因启动子,大小为300bp,活性较高,它启动基因表达的能力约为人的骨骼肌α-actin启动子与肌酸激酶启动子的2倍左右;CMV-Myo Gpro是Myo G基因启动子之前连接了一段CMV增强子序列,在C2C12细胞中CMV-Myo Gpro复合启动子的转录活性较牛Myo G启动子提高了9倍左右,达到了强启动子CMV启动子的72%,在牛骨骼肌卫星细胞中,CMV增强子提高转录活性的效果更明显;Myo Gpro-double是含有两个拷贝数调控元件的Myo G启动子片段,其转录活性显著高于普通Myo G基因启动子,约为Myo G基因启动子转录活性的3.8倍,三种启动子都具有较高的活性和较好的肌肉特异性。本研究获得了含上述三种有肌肉特异性启动子的IGF2真核表达载体,通过细胞转染及IGF2表达量检测,筛选出具有较高活性和较强肌肉特异性的IGF2表达载体,并探索其对牛骨骼肌卫星细胞增殖的调控作用。主要研究结果如下:(1)肌肉特异性的IGF2表达载体的构建本实验根据Gene Bank中公布的牛IGF2基因序列,设计引物,应用PCR方法,以MDSCs基因组DNA为模板扩增牛IGF2基因CDS序列,并利用实验室前期构建的desminpro,CMV-Myo Gpro,Myo Gpro-Double三种肌肉特异性启动子构建了p GL3-desminpro-IGF2,p GL3-CMV-Myo Gpro-IGF2,p GL3-Myo Gpro-Double-IGF2三种带有肌肉特异性启动子的IGF2真核表达载体;(2)表达载体活性与特异性的筛选瞬时转染MDSCs和牛胎儿成纤维细胞,RT-PCR检测比较三种启动子启动IGF2基因在肌肉中的表达效率,结果表明p GL3-desminpro-IGF2是相对高效,并具有肌肉特异性的表达载体;(3)Ed U检测p GL3-desminpro-IGF2转入细胞后对MDSCs增殖的影响p GL3-desminpro-IGF2载体转入MDSCs后,EDU检测证明p GL3-desminpro-IGF2能够提高细胞增殖率,与对照相比,增殖率为27.31%;(4)检测p GL3-desminpro-IGF2转入细胞后细胞内相关基因的表达变化RT-PCR检测p GL3-desminpro-IGF2转染后细胞周期相关基因的表达变化发现IGF2是通过下调CDKs蛋白抑制因子P27的表达,上调CDK6的表达而加速细胞增殖的;为了进一步研究IGF2的作用机理,我们通过向MDSCs中添加生长因子IGF2作用后进行Western blot检测,结果表明该基因可能是通过调节AKT磷酸化过程而加速细胞增殖的。本研究获得了能够在MDSCs中高效特异性表达的真核表达载体p GL3-desminpro-IGF2,其在MDSCs中能够高效特异性表达并能显著提高成肌细胞的增殖率,为生产肌肉产量高的转基因肉牛奠定了基础。
[Abstract]:IGF2 (insulin-like growth factor 2) is a protein. It is composed of 67 amino acid residues. It plays an important role in the growth and development of the individual. It is an important regulatory factor for the growth and development of skeletal muscle. In recent years, the research on IGF2 has been deepened, but the high quality and fast growth beef cattle are developed by means of IGF2 expression. The purpose of this study is to obtain a IGF2 expression vector with highly specific promoter and to study its effect on the proliferation of bovine skeletal muscle satellite cells (MDSCs). In order to improve the expression efficiency of IGF2 in the muscle tissue, it is necessary to obtain a high expression vector containing IGF2, in which the selection of promoter is a one. Important factors. At present, cytomegalovirus (CMV) promoter is one of the frequently selected promoters of exogenous gene expression in eukaryotic cells. The promoter is very efficient, but it is almost no tissue specific. It can not be used in the production of transgenic beef cattle for the safety of genetically modified organisms. The promoter is necessary for the expression of foreign genes in eukaryotic cells. This study applies three promoters with specific and relatively high activity in the early laboratory: Desminpro (300) is the promoter of the bovine nodin gene, the size is 300bp, the activity is high, and its ability to start the gene expression is about human skeletal muscle alpha -actin The promoter is about 2 times that of the creatine kinase promoter; CMV-Myo Gpro is a CMV enhancer sequence connected before the promoter of the Myo G gene. The transcriptional activity of the CMV-Myo Gpro complex promoter in C2C12 cells is increased about 9 times more than that of the bovine Myo G promoter, reaching 72% of the strong promoter CMV initiator. In the bovine skeletal muscle satellite cells, CMV increases. The effect of the hadron to enhance the transcriptional activity is more obvious; Myo Gpro-double is a Myo G promoter fragment containing two copies of the regulatory element, and its transcriptional activity is significantly higher than that of the common Myo G gene promoter, about 3.8 times the transcriptional activity of the Myo G gene promoter, and the three promoters have higher activity and better muscle specificity. The IGF2 eukaryotic expression vector containing three kinds of muscle specific promoters was obtained. Through cell transfection and IGF2 expression detection, the IGF2 expression vector with high activity and strong muscle specificity was screened and its regulation effect on the proliferation of bovine skeletal muscle satellite cells was explored. The main results are as follows: (1) muscle specific IGF2 The construction of the expression vector based on the sequence of bovine IGF2 gene published in Gene Bank, designed primers, applied the PCR method, amplified the CDS sequence of bovine IGF2 gene with the MDSCs genome DNA as a template, and constructed the three kinds of muscle specific promoters, which were constructed in the early stage of the laboratory, desminpro, CMV-Myo Gpro, Myo. 3-CMV-Myo Gpro-IGF2, P GL3-Myo Gpro-Double-IGF2 three kinds of IGF2 eukaryotic expression vectors with muscle specific promoters; (2) transient transfection of MDSCs and bovine fetal fibroblasts by screening the activity and specificity of the expression vector. RT-PCR detection compared the expression efficiency of the IGF2 gene in the muscle by three promoters, and the results showed P GL3-desminpro. -IGF2 is relatively efficient and has a specific expression vector of muscle specificity; (3) Ed U tests the effect of P GL3-desminpro-IGF2 on the proliferation of MDSCs after the transfer of P GL3-desminpro-IGF2 vector into MDSCs, EDU tests show that P GL3-desminpro-IGF2 can increase the cell proliferation rate, and the rate of proliferation is 27.31% compared with that of the camera. (4) detection 2 changes in the expression of related genes after transfection of cells RT-PCR detected changes in the expression of cell cycle related genes after P GL3-desminpro-IGF2 transfection found that IGF2 was regulated by down regulation of the expression of the CDKs protein inhibitory factor P27 to up regulate the expression of CDK6 and accelerate the proliferation of cells. In order to further study the mechanism of IGF2, we pass to MDSCs. Western blot was detected by adding growth factor IGF2. The results showed that the gene may be accelerated by the regulation of AKT phosphorylation. This study obtained the eukaryotic expression vector, P GL3-desminpro-IGF2, which was highly expressed in MDSCs, which could be highly expressed in MDSCs and could be significantly extracted in MDSCs. The proliferation rate of high myoblasts lays the foundation for producing transgenic beef cattle with high muscle production.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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