本文選題：IGF2 + 肌肉特異性啟動(dòng)子�。� 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：IGF2(胰島素樣生長因子2)是一種蛋白質(zhì),它由67個(gè)氨基酸殘基組成,在個(gè)體的生長發(fā)育過程中起重要作用,是骨骼肌生長發(fā)育的重要調(diào)控因子。近年來,對IGF2的研究逐漸深入,但是通過過表達(dá)IGF2的方式來培育高產(chǎn)、生長發(fā)育快的優(yōu)質(zhì)肉牛方面的報(bào)道較少。本研究旨在獲得帶有高效特異性啟動(dòng)子的IGF2表達(dá)載體,研究其對牛骨骼肌衛(wèi)星細(xì)胞(簡稱MDSCs)增殖的影響。欲提高IGF2在肌肉組織中的表達(dá)效率,首先需要獲得一個(gè)含有IGF2的高效表達(dá)的載體,其中啟動(dòng)子的選擇是一個(gè)重要因素。目前,巨細(xì)胞病毒(CMV)啟動(dòng)子是外源基因在真核細(xì)胞中表達(dá)常選用的啟動(dòng)子之一,該啟動(dòng)子非常高效,但其幾乎不具有組織特異性,出于轉(zhuǎn)基因生物安全方面的考慮,不能將其應(yīng)用于轉(zhuǎn)基因肉牛的生產(chǎn)中。所以,選取特異高效的啟動(dòng)子對于外源基因在真核細(xì)胞中的表達(dá)是必要的,本研究應(yīng)用的是實(shí)驗(yàn)室前期構(gòu)建的具有特異性且活性相對較高的三種啟動(dòng)子:Desminpro(300)是牛結(jié)蛋白基因啟動(dòng)子,大小為300bp,活性較高,它啟動(dòng)基因表達(dá)的能力約為人的骨骼肌α-actin啟動(dòng)子與肌酸激酶啟動(dòng)子的2倍左右;CMV-Myo Gpro是Myo G基因啟動(dòng)子之前連接了一段CMV增強(qiáng)子序列,在C2C12細(xì)胞中CMV-Myo Gpro復(fù)合啟動(dòng)子的轉(zhuǎn)錄活性較牛Myo G啟動(dòng)子提高了9倍左右,達(dá)到了強(qiáng)啟動(dòng)子CMV啟動(dòng)子的72%,在牛骨骼肌衛(wèi)星細(xì)胞中,CMV增強(qiáng)子提高轉(zhuǎn)錄活性的效果更明顯;Myo Gpro-double是含有兩個(gè)拷貝數(shù)調(diào)控元件的Myo G啟動(dòng)子片段,其轉(zhuǎn)錄活性顯著高于普通Myo G基因啟動(dòng)子,約為Myo G基因啟動(dòng)子轉(zhuǎn)錄活性的3.8倍,三種啟動(dòng)子都具有較高的活性和較好的肌肉特異性。本研究獲得了含上述三種有肌肉特異性啟動(dòng)子的IGF2真核表達(dá)載體,通過細(xì)胞轉(zhuǎn)染及IGF2表達(dá)量檢測,篩選出具有較高活性和較強(qiáng)肌肉特異性的IGF2表達(dá)載體,并探索其對牛骨骼肌衛(wèi)星細(xì)胞增殖的調(diào)控作用。主要研究結(jié)果如下:(1)肌肉特異性的IGF2表達(dá)載體的構(gòu)建本實(shí)驗(yàn)根據(jù)Gene Bank中公布的牛IGF2基因序列,設(shè)計(jì)引物,應(yīng)用PCR方法,以MDSCs基因組DNA為模板擴(kuò)增牛IGF2基因CDS序列,并利用實(shí)驗(yàn)室前期構(gòu)建的desminpro,CMV-Myo Gpro,Myo Gpro-Double三種肌肉特異性啟動(dòng)子構(gòu)建了p GL3-desminpro-IGF2,p GL3-CMV-Myo Gpro-IGF2,p GL3-Myo Gpro-Double-IGF2三種帶有肌肉特異性啟動(dòng)子的IGF2真核表達(dá)載體;(2)表達(dá)載體活性與特異性的篩選瞬時(shí)轉(zhuǎn)染MDSCs和牛胎兒成纖維細(xì)胞,RT-PCR檢測比較三種啟動(dòng)子啟動(dòng)IGF2基因在肌肉中的表達(dá)效率,結(jié)果表明p GL3-desminpro-IGF2是相對高效,并具有肌肉特異性的表達(dá)載體;(3)Ed U檢測p GL3-desminpro-IGF2轉(zhuǎn)入細(xì)胞后對MDSCs增殖的影響p GL3-desminpro-IGF2載體轉(zhuǎn)入MDSCs后,EDU檢測證明p GL3-desminpro-IGF2能夠提高細(xì)胞增殖率,與對照相比,增殖率為27.31%;(4)檢測p GL3-desminpro-IGF2轉(zhuǎn)入細(xì)胞后細(xì)胞內(nèi)相關(guān)基因的表達(dá)變化RT-PCR檢測p GL3-desminpro-IGF2轉(zhuǎn)染后細(xì)胞周期相關(guān)基因的表達(dá)變化發(fā)現(xiàn)IGF2是通過下調(diào)CDKs蛋白抑制因子P27的表達(dá),上調(diào)CDK6的表達(dá)而加速細(xì)胞增殖的;為了進(jìn)一步研究IGF2的作用機(jī)理,我們通過向MDSCs中添加生長因子IGF2作用后進(jìn)行Western blot檢測,結(jié)果表明該基因可能是通過調(diào)節(jié)AKT磷酸化過程而加速細(xì)胞增殖的。本研究獲得了能夠在MDSCs中高效特異性表達(dá)的真核表達(dá)載體p GL3-desminpro-IGF2,其在MDSCs中能夠高效特異性表達(dá)并能顯著提高成肌細(xì)胞的增殖率,為生產(chǎn)肌肉產(chǎn)量高的轉(zhuǎn)基因肉牛奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823

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本文編號(hào)：1832061